CN111566205A

CN111566205A - Cell population comprising adherent stem cells, method for producing same, and pharmaceutical composition

Info

Publication number: CN111566205A
Application number: CN201880084424.7A
Authority: CN
Inventors: 稻生溪太; 小林千穗; 山口翔; 梅田伸好
Original assignee: Kaneka Corp
Current assignee: Kaneka Corp
Priority date: 2017-12-28
Filing date: 2018-12-28
Publication date: 2020-08-21
Also published as: EP3733838A1; EP3733838A4; WO2019132025A1; JPWO2019132025A1; US20200360444A1

Abstract

The present invention addresses the problem of providing a cell population that contains adherent stem cells that exhibit high immunosuppressive activity and/or proliferation properties, a method for producing the same, and a pharmaceutical composition that contains the cell population. According to the present invention, there can be provided a method for producing a cell population comprising adherent stem cells, the method comprising: a cell population having the cell characteristics of (a) positive for the expression of the PLIN2 gene and NEFM gene and (b) 1.50 or more in terms of the relative expression level of the PLIN2 gene to the expression level of the SDHA gene is obtained.

Description

Cell population comprising adherent stem cells, method for producing same, and pharmaceutical composition

Technical Field

The present invention relates to a method for producing a cell population containing adherent stem cells such as mesenchymal stem cells. In addition, the present invention relates to a cell population comprising adherent stem cells, and a pharmaceutical composition. The present invention also relates to a method for monitoring immunosuppressive action and/or proliferation of adherent stem cells, a method for evaluating a donor and/or a biological sample collected from the donor, and a method for judging and/or predicting optimal enzyme treatment conditions, which utilize an index relating to a cell population containing adherent stem cells.

Background

Adherent stem cells such as Mesenchymal stem cells, also called Mesenchymal stromal cells (mesenchymeal stromal cells), are adult stem cells reported to be present in bone marrow, adipose tissue, dental pulp, and the like, and have recently been found to be present in fetal appendages such as placenta, umbilical cord, fetal membrane, and the like.

Since the adherent stem cells have immunosuppressive ability, it has been reported that acute Graft Versus Host Disease (GVHD), crohn's disease which is an inflammatory bowel disease, and the like can be treated by intravenously administering a cell preparation containing mesenchymal stem cells.

Patent document 1 describes a method for producing an amniotic mesenchymal cell composition, a method for cryopreservation, and a therapeutic agent. Specifically, it is described that amnion mesenchyme cells can be prepared into a cell preparation optimized for transplantation by freezing and preserving a mixture containing amnion mesenchyme cells in a solution containing 5 to 10 mass% of dimethyl sulfoxide and 5 to 10 mass% of hydroxyethyl starch or 1 to 5 mass% of dextran.

Patent document 2 describes a method for producing a cell population containing mesenchymal stem cells, which comprises a screening step of screening mesenchymal stem cells having a relatively high proliferation potency by treating a cell population containing mesenchymal stem cells having different proliferation potency with a physical stimulus or a chemical stimulus, wherein the screened mesenchymal stem cells having a relatively high proliferation potency are CD106 negative, and the expression level of a metallothionein family gene is increased as compared with that before the treatment with the physical stimulus or the chemical stimulus.

Patent document 3 describes a method for producing a population of amniotic mesenchymal stem cells, which comprises: (D) collecting a cell population of mesenchymal cells from an amniotic membrane of a mammal; (E) at 400-35000/cm²Inoculating the collected cell population at the cell concentration of (2) and performing initial culture for 2 to 3 days; (F) inoculating the cells at a cell concentration of 1/5000 or more and less than 1/10 in the initial culture, subculturing the cells with medium replacement of 2 times for 1 week, and repeating the subculturing step 3 to 4 times; and (G) a step of, when a cell colony of cells having a spindle-shaped morphology is formed in the subculture, maintaining the culture of the cells in the same culture dish until the cells reach confluence.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open publication No. 2015-61520

Patent document 2: international publication WO2017/073656

Patent document 3: international publication WO2013/077428

Disclosure of Invention

Problems to be solved by the invention

In recent years, with the rise in medical costs, there has been an active trend toward the medical economy of expensive pharmaceuticals, such as biopharmaceuticals. In order to realize and popularize a cell preparation containing living cells as an active ingredient, it is important to reduce the production cost of the cell preparation.

Among them, in recent years, it has been found that an adherent stem cell population is a heterogeneous cell population including various cells having different differentiation ability, proliferation ability, and cytokine production ability. In order to reduce the manufacturing cost of cell preparations, it is necessary to efficiently produce highly functional adherent stem cells, in particular, adherent stem cell populations exhibiting high therapeutic effects and high proliferation.

Patent document 1 describes that a mixture containing amniotic mesenchymal cells can be cryopreserved in a specific cryopreservation solution, thereby suppressing a decrease in the survival rate of the amniotic mesenchymal cells after thawing, and the cryopreserved amniotic mesenchymal cells can be prepared into a cell preparation optimized for transplantation. In addition, patent document 2 describes that mesenchymal stem cells having a relatively high proliferation capacity can be prepared by treatment with physical stimulation or chemical stimulation. However, patent documents 1 and 2 do not describe or teach the selective production of mesenchymal stem cells having specific excellent characteristics from mesenchymal stem cells, specifically, the selective production of a large amount of cell populations containing mesenchymal stem cells exhibiting high immunosuppressive activity and proliferative property using the characteristics of mesenchymal stem cells as an index. Even when the production methods described in patent documents 1 and 2 are used, the immunosuppressive action and specific proliferation rate of the obtained cells are insufficient, and therefore, not only is the time and material cost required for cell culture consumed, but also the drug effect of the cell preparation can be improved only by containing a large amount of cells as an active ingredient, and therefore, it is difficult to produce the cell preparation at low cost.

Patent document 3 does not describe nor teach the selection of a large amount of cell populations containing mesenchymal stem cells exhibiting high immunosuppressive activity and proliferative activity, using the characteristics of mesenchymal stem cells contained in the mesenchymal stem cell population as an index, although a mesenchymal stem cell population having high proliferative and differentiative capacities is prepared by seeding cells at a low density. In addition, the mesenchymal stem cell population described in patent document 3 has insufficient immunosuppressive action and specific proliferation rate. Actually, the present inventors have further studied the production method described in patent document 3, and as a result, have confirmed that the process of seeding at a low density and repeating subculture 3 to 4 times requires not only 23 days but also a specific growth rate of the obtained mesenchymal stem cell population of 0.34(1/day) is insufficient. Therefore, even when the production method described in patent document 3 is used, it is impossible to shorten the cell culture time, and it is difficult to produce a cell preparation at low cost.

The present invention addresses the problem of providing a cell population comprising adherent stem cells that exhibit high immunosuppressive activity and/or proliferation properties, a method for producing the cell population, and a pharmaceutical composition comprising the cell population, which are useful for reducing the production cost of a cell preparation. Further, another object of the present invention is to provide a method for monitoring immunosuppressive action and/or proliferation of adherent stem cells, a method for evaluating a donor and/or a biological sample collected from the donor, and a method for judging and/or predicting optimal enzyme treatment conditions, using an index relating to a cell population including the adherent stem cells.

Means for solving the problems

As a result of intensive studies to solve the above problems, the present inventors have found that adherent stem cells in which the expression of the PLIN2 gene and the NEFM gene is positive and the relative expression level of the PLIN2 gene to the expression level of the SDHA gene is 1.50 or more are contained in a cell population containing the adherent stem cells, and that a cell population containing adherent stem cells having the above cell characteristics exhibits a high immunosuppressive action. The present inventors have also found that a cell population containing adherent stem cells having the above-described cell characteristics has a high specific growth rate. The present inventors have also found that the immunosuppressive action and/or proliferation of adherent stem cells can be monitored by using, as an indicator, a cell population containing adherent stem cells in which the expression of the PLIN2 gene and NEFM gene is positive and the relative expression level of the PLIN2 gene with respect to the expression level of the SDHA gene is 1.50 or more. Further, it has been found that the quality of a donor and/or a biological sample collected from the donor can be evaluated from the viewpoint of efficiently obtaining adherent stem cells exhibiting high immunosuppressive action and/or proliferation. Further, it has been found that the optimal enzyme treatment conditions for a biological sample collected from a donor can be judged and/or predicted from the viewpoint of efficiently obtaining adherent stem cells exhibiting high immunosuppressive action and/or proliferation. The present invention has been completed based on these findings.

That is, the present specification can provide the following invention.

[1] A method of making a population of cells comprising adherent stem cells, the method comprising: obtaining a cell population having the cell characteristics of (a) and (b) shown below,

(a) the cell population was positive for expression of the PLIN2 gene and the NEFM gene,

(b) the relative expression level of the PLIN2 gene relative to the expression level of the SDHA gene in the cell population is 1.50 or more.

[2] The production method according to [1], wherein the relative expression amount of the NEFM gene of the cell population to the expression amount of the SDHA gene is less than 0.02.

[3] The production method according to [1] or [2], wherein the relative expression level of the PLIN2 gene in the cell population with respect to the expression level of the NEFM gene is 250 or more.

[4] A method of producing a cell population comprising mesenchymal stem cells, the method comprising: obtaining a cell population having the cell characteristics of (a) and (b) shown below,

(a) the relative expression amount of the PLIN2 gene relative to the expression amount of the SDHA gene of the cell population is more than 0.30, and

(b) the relative expression level of the NEFM gene of the cell population relative to the expression level of the SDHA gene is less than 1.50.

[5] A cell population comprising adherent stem cells, which is a cell population having the cell characteristics of (a) and (b) shown below,

[6] The cell population according to [5], wherein the relative expression amount of the NEFM gene relative to the expression amount of the SDHA gene of the cell population is less than 0.02.

[7] The cell population according to [5] or [6], wherein the relative expression amount of the PLIN2 gene relative to the expression amount of the NEFM gene in the cell population is 250 or more.

[8] A cell population comprising mesenchymal stem cells, which is a cell population having the cell characteristics of (a) and (b) shown below,

[9] The cell population according to any one of [5] to [8], wherein the adherent stem cells are derived from a fetal appendage.

[10] A pharmaceutical composition comprising the cell population of any one of [5] to [9], and a pharmaceutically acceptable vehicle.

[11] A pharmaceutical composition comprising the cell population according to any one of [5] to [9] and another administrable cell.

[12]According to [10]]Or [11]The pharmaceutical composition, wherein the 1 dose of the human adherent stem cells is 1 × 10¹²Less than one/kg body weight.

[13] The pharmaceutical composition according to any one of [10] to [12], wherein the pharmaceutical composition is an injectable preparation.

[14] The pharmaceutical composition according to any one of [10] to [12], wherein the pharmaceutical composition is a preparation for transplantation having a cell mass or a sheet-like structure.

[15] The pharmaceutical composition according to any one of [10] to [14], which is a therapeutic agent for an immune-related disease.

[16] The cell population according to any one of [5] to [9], wherein the specific growth rate is 0.40(1/day) or more.

[17] The cell population according to any one of [5] to [9], wherein the relative expression level of TNFAIP6 gene in the cell population with respect to the expression level of SDHA gene is 0.5 or more.

[18] A cell population obtained by the production method according to any one of [1] to [4 ].

[18-2] the production method according to any one of [1] to [4], wherein the relative expression level of TNFAIP6 gene in the cell population with respect to the expression level of SDHA gene is 0.5 or more.

[19] Use of the cell population according to any one of [5] to [9] for the production of a pharmaceutical composition.

[20]According to [19]]The use of (1), wherein the pharmaceutical composition is 1 dose of 1 × 10 to human adherent stem cells¹²A pharmaceutical composition for treating or preventing obesity per kg body weight.

[21] The use according to [19] or [20], wherein the pharmaceutical composition is an injectable preparation.

[22] The use according to [19] or [20], wherein the pharmaceutical composition is a preparation for transplantation of a cell pellet or a sheet-like structure.

[23] The use according to any one of [19] to [22], wherein the pharmaceutical composition is a therapeutic agent for an immune-related disease.

[24] The cell population according to any one of [5] to [9], which is used for the treatment of a disease.

[25]According to [24]]The cell population, wherein 1 dose of the human adherent stem cells is 1 × 10¹²Less than one/kg body weight.

[26] The cell population according to [24] or [25], which is an injectable preparation.

[27] The cell population according to [24] or [25], which is a preparation for transplantation of a cell pellet or a sheet-like structure.

[28] The cell population according to any one of [24] to [27], wherein the disease is an immune-related disease.

[29] A method of treating a disease, the method comprising: administering to a patient or subject in need of treatment a cell population according to any one of [5] to [9 ].

[30]According to [29]]The method for treating a disease, wherein the dose of 1 to human adherent stem cells is 1 × 10¹²Less than one/kg body weight.

[31] The method for treating a disease according to [29] or [30], which is an injectable preparation.

[32] The method for treating a disease according to [29] or [30], which is a preparation for transplantation of a cell pellet or a sheet-like structure.

[33] The method for treating a disease according to any one of [29] to [32], wherein the disease is an immune-related disease.

[34] A composition comprising the cell population of any one of [5] to [9], and a medium.

[35] A method of monitoring immunosuppression and/or proliferation of adherent stem cells, the method comprising:

the presence or absence of the expression of the PLIN2 gene and NEFM gene, and the relative expression level of the PLIN2 gene with respect to the expression level of the SDHA gene were measured in a cell population including adherent stem cells, and the cell characteristics of (a) and (b) shown below were used as indices,

[36] A method of monitoring the proliferative and/or immunosuppressive effects of mesenchymal stem cells, the method comprising:

in a cell population containing mesenchymal stem cells, the relative expression level of PLIN2 gene relative to the expression level of SDHA gene and the relative expression level of NEFM gene relative to the expression level of SDHA gene were measured, and the cell characteristics of (a) and (b) shown below were used as indices,

[37] A method of evaluating a donor and/or a biological sample collected from a donor, the method comprising:

a cell population containing adherent stem cells was collected from a donor, the presence or absence of the expression of the PLIN2 gene and NEFM gene, and the relative expression level of the PLIN2 gene with respect to the expression level of the SDHA gene were measured, and the cell characteristics of (a) and (b) shown below were used as indices for evaluation,

[38] A method of evaluating a donor and/or a biological sample collected from a donor, the method comprising:

a cell population containing mesenchymal stem cells was collected from a donor, and the relative expression level of the PLIN2 gene with respect to the expression level of the SDHA gene and the relative expression level of the NEFM gene with respect to the expression level of the SDHA gene were measured and evaluated using the cell characteristics of (a) and (b) shown below as indices,

[39] A method of determining and/or predicting optimal enzyme treatment conditions for a sample of a biological organism, the method comprising:

the presence or absence of the expression of the PLIN2 gene and NEFM gene, and the relative expression level of the PLIN2 gene with respect to the expression level of the SDHA gene were measured for a cell population obtained by subjecting a biological sample collected from a donor to an enzyme treatment, and evaluated using the cell characteristics of (a) and (b) shown below as an index,

[40] A method for determining and/or predicting optimal enzyme treatment conditions for a biological sample, the method comprising:

the relative expression level of PLIN2 gene with respect to the expression level of SDHA gene and the relative expression level of NEFM gene with respect to the expression level of SDHA gene were measured for a cell population obtained by subjecting a biological sample collected from a donor to an enzyme treatment, and evaluated using the cell characteristics of (a) and (b) shown below as an index,

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, a cell population comprising adherent stem cells exhibiting a high immunosuppressive effect can be obtained. In addition, according to the present invention, a cell population comprising adherent stem cells exhibiting high proliferation can be obtained. In addition, according to the present invention, a method for monitoring hyperimmune suppression and/or proliferation in a cell population comprising adherent stem cells over time can be provided. In addition, according to the present invention, as an index for formation of a cell population including adherent stem cells exhibiting high immunosuppressive action and/or proliferation properties, the relative expression amounts of various genes with respect to housekeeping genes can be used, whereby a cell preparation (pharmaceutical composition) can be produced at low cost.

Drawings

Fig. 1 shows the results of survival rate of example 2.

Fig. 2 shows the results of survival rate of example 7.

Detailed Description

The following description will specifically describe embodiments of the present invention, but the following description is for easy understanding of the present invention, and the scope of the present invention is not limited to the following embodiments, and other embodiments obtained by appropriately replacing the configurations of the following embodiments by those skilled in the art are also included in the scope of the present invention.

[1] Description of the words

The term "fetal appendages" as used herein refers to the fetal membranes, placenta, umbilical cord and amniotic fluid. The term "fetal membrane" refers to a fetal sac containing amniotic fluid of a fetus and is formed from an amniotic membrane, a chorion, and an decidua membrane from the inside. Wherein the amniotic membrane and chorion originate from the fetus. By "amniotic membrane" is meant a blood vessel-deficient transparent membrane located on the innermost layer of the fetal membrane. The inner layer of the amniotic membrane (also referred to as epithelial cell layer) is coated with a layer of epithelial cells having a secretion function to secrete amniotic fluid, and the outer layer of the amniotic membrane (also referred to as extracellular matrix layer, corresponding to stroma) contains adherent stem cells.

The term "adherent stem cell" as used herein refers to a stem cell satisfying the following definition, "Mesenchymal stromal cells (mesenchyme stromal cells)" and "Mesenchymal stem cells (mesenchyme stem cells)" are also included in the adherent stem cell. In the present specification, "mesenchymal stem cell" is sometimes also referred to as "MSC".

Among adult stem cells (tissue stem cells) that can be collected from various tissues and organs, cells satisfying the following definition can be used as "adherent stem cells". Examples of the adult stem cells (tissue stem cells) include: bone marrow-derived mesenchymal stem cells, hematopoietic stem cells, stem cells in umbilical cord blood, umbilical cord-derived stem cells, amnion-derived stem cells, amniotic fluid stem cells, placental villus cell-derived mesenchymal stem cells, neural stem cells, adipose tissue-derived stem cells, pancreatic stem cells, synovial mesenchymal stem cells, dental pulp-derived stem cells derived from deciduous teeth, sperm stem cells (GS cells), testicular pluripotent stem cells (mGS cells), corneal epithelial stem cells, corneal parenchymal stem cells, pigment stem cells, tissue stem cells in organs, and the like, but are not particularly limited.

Definition of adherent Stem cells

i) Adherence to plastic was shown under culture conditions in standard medium.

ii) surface antigens CD73, CD90 are positive and CD326 is negative.

The term "adherent stem cell population" as used herein refers to a cell population including adherent stem cells, and the form thereof is not particularly limited, and examples thereof include: cell pellet, cell sheet, cell aggregate, cell float, cell suspension, or the like.

The "adherent stem cells" may satisfy the above definition, and the presence or absence of differentiation into bone, cartilage, fat, or the like is not particularly limited. For example, "adherent stem cells" in the present specification also include cells having the ability to differentiate into bone, cartilage, and fat, such as mesenchymal stem cells. The "adherent stem cells" also include cells that, although satisfying the above definition, do not have the ability to differentiate into bone, cartilage, fat, and the like. In addition, the "adherent stem cells" also include cells that, although satisfying the above definition, differentiate into only any 1 or 2 of bone, cartilage, and fat.

The term "amnion adherent stem cell" as used herein refers to an amnion-derived adherent stem cell, and can be used without distinction from "amnion mesenchymal stromal cell". In the present specification, "amniotic mesenchymal stem cells" are sometimes referred to as "amniotic MSCs".

The "ratio of adherent stem cells positive for a surface antigen" in the present specification means a ratio of cells positive for a surface antigen obtained by flow cytometry analysis as described in examples described later. In the present specification, the "ratio of adherent stem cells positive for a surface antigen" is sometimes referred to as "positive rate".

The "ratio of adherent stem cells negative for surface antigen" in the present specification means a ratio of cells negative for surface antigen obtained by flow cytometry analysis as described in examples described later. In the present specification, the "ratio of adherent stem cells negative for surface antigens" is sometimes referred to as "negative rate".

The term "proliferation ability" as used herein refers to the ability of a cell to undergo cell division, thereby increasing the number of cells. In the present specification, "high proliferation potency" can be used without distinction from "high proliferation potency". The proliferative capacity of an adherent stem cell population can be evaluated using the number of cells obtained per 1 culture batch, specific proliferation rate, number of doublings, doubling time, and/or number of passages. The method for measuring the specific growth rate is as described later in this specification.

The "number of cells obtained per 1 culture batch" in the present specification means the number of cells obtained per unit surface area and per unit number of culture days of the culture vessel in 1 culture. Thus, the unit of "the number of cells obtained per 1 culture batch" is (one/cm)²/day)。

The term "immunosuppressive action" as used herein refers to an action of suppressing an immune response of an organism. In the present specification, "immunosuppressive action" can be used without distinction from "immunosuppressive ability". The immunosuppressive effect of the adherent stem cell population can be evaluated by using the expression level of TNFAIP6 (tumor necrosis factor, alpha-induced protein 6) gene, the proliferation inhibition rate of human peripheral blood mononuclear cells (hPBMC) in a mixed lymphocyte reaction test (MLR test), and animal experiments. The method for evaluating the immunosuppressive effect is as described later in the present specification.

[2] Cell populations comprising adherent stem cells

The cell population comprising adherent stem cells provided by the invention is characterized in that:

(a) the expression of PLIN2 gene and NEFM gene in the above cell population was positive,

(b) the relative expression level of PLIN2 gene relative to the expression level of SDHA gene in the cell population is 1.50 or more.

The cell population containing adherent stem cells provided by the present invention forms a cell population containing adherent stem cells that exhibit high immunosuppressive activity and/or proliferation when (a) the expression of the PLIN2 gene and NEFM gene of the cell population is positive and (b) the relative expression level of the PLIN2 gene of the cell population with respect to the expression level of the SDHA gene is 1.50 or more. Therefore, in the present invention, the above conditions can be used as an index for forming a cell population exhibiting a high immunosuppressive action and/or proliferation. Furthermore, by measuring the above-mentioned index over time, the change in the immunosuppressive action and/or the proliferation of the adherent stem cells can be quickly grasped and predicted. In addition, according to the present invention, by using the above-mentioned index, the quality of the donor itself and/or the biological sample collected from the donor can be evaluated. Further, according to the present invention, by using the above-mentioned index, it is possible to judge and/or predict whether or not an enzyme treatment method is suitable when a biological sample collected from a donor is subjected to an enzyme treatment.

PLIN2 refers to Perilipin 2. The sequence of the PLIN2 gene was registered in the gene database of the National Center for Biotechnology Information (National Center for Biotechnology Information) as ID: 123. PLIN2 is a gene having a nucleotide sequence represented by SEQ ID NO. 1 or a gene encoding a polypeptide having an amino acid sequence represented by SEQ ID NO. 2.

NEFM refers to the sequence of the NeFM gene, which is registered as ID: 4741 in the gene database of the national center for Biotechnology information, NEFM is a gene consisting of the base sequence shown in SEQ ID NO. 3, or a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 4.

SDHA is a member of housekeeping genes, and refers to the succinate dehydrogenase complex, subunit A. The sequence of the SDHA gene is registered in the gene database of the national center for Biotechnology information as ID: 6389. SDHA is a gene having a base sequence represented by SEQ ID NO. 5 or a gene encoding a polypeptide having an amino acid sequence represented by SEQ ID NO. 6.

The upper limit of the relative expression level of the PLIN2 gene with respect to the expression level of the SDHA gene may be, for example, 20.0 or less, 19.0 or less, 18.0 or less, 17.0 or less, 16.0 or less, 15.0 or less, 14.0 or less, 13.0 or less, 12.0 or less, 11.0 or less, 10.0 or less, 9.0 or less, 8.9 or less, 8.8 or less, 8.7 or less, 8.6 or less, 8.5 or less, 8.4 or less, 8.3 or less, 8.2 or less, 8.1 or less, 8.0 or less, 7.99 or less, 7.98 or less, 7.97 or less, 7.96 or less, 7.95 or less, 7.94 or less, 7.8 or less, 7.7 or less, 7.6 or less, 7.5 or less, 7.4 or less, 7.3 or less, 7.2 or less, 7.1 or less, 7.0 or less, 6.9 or less, 6.8 or less, 6.7 or less, 6.6 or less, 6.5 or less, 6.4 or less, 6.3 or less, 6.2 or less, 6.1 or less, 6.0 or less, 5.9 or less, 5.8 or less, 5.7 or less, 5.6 or less, 5.5 or less, 5.4 or less, 5.3 or less, 5.2 or less, 5.1 or less, or 5.0 or less.

The lower limit of the relative expression amount of the PLIN2 gene with respect to the expression amount of the SDHA gene may be, for example, 1.51 or more, 1.52 or more, 1.53 or more, 1.54 or more, 1.55 or more, 1.56 or more, 1.57 or more, 1.58 or more, 1.59 or more, 1.60 or more, 1.61 or more, 1.62 or more, 1.63 or more, 1.64 or more, 1.65 or more, 1.66 or more, 1.67 or more, 1.68 or more, 1.69 or more, 1.70 or more, 1.71 or more, 1.72 or more, 1.73 or more, 1.74 or more, 1.75 or more, 1.76 or more, 1.77 or more, 1.78 or more, 1.79 or more, 1.80 or more, 1.81 or more, 1.82 or more, 1.83 or more, 1.84 or more, 1.85 or more, 1.86 or more, 1.87 or more, 1.88 or more, 1.89 or more, 1.90 or more, 1.91 or more, 1.92 or more, 2.93 or more, 2.6 or more, 2.93 or more, 2.3.93 or more, 2.93 or more, 2.3., 3.4 or more, 3.5 or more, 3.6 or more, 3.7 or more, 3.8 or more, 3.9 or more, 4.0 or more, 4.1 or more, 4.2 or more, 4.3 or more, 4.4 or more, 4.5 or more, 4.6 or more, 4.7 or more, 4.8 or more, 4.9 or more, or 5.0 or more.

The upper limit of the relative expression amount of the NEFM gene with respect to the expression amount of the SDHA gene may be, for example, less than 0.020, 0.019 or less, 0.018 or less, 0.017 or less, 0.016 or less, 0.015 or less, 0.014 or less, 0.013 or less, 0.012 or less, 0.011 or less, 0.010 or less, 0.009 or less, or 0.008 or less.

The lower limit of the relative expression level of the NEFM gene with respect to the expression level of the SDHA gene may be, for example, 0.0001 or more, 0.0002 or more, 0.0003 or more, 0.0004 or more, 0.0005 or more, 0.0006 or more, 0.0007 or more, 0.0008 or more, 0.0009 or more, 0.001 or more, 0.002 or more, 0.003 or more, 0.004 or more, 0.005 or more, 0.006 or more, 0.007 or more, or 0.008 or more.

The upper limit of the relative expression amount of the PLIN2 gene with respect to the expression amount of the NEFM gene may be, for example, 40000 or less, 45000 or less, 30000 or less, 25000 or less, 20000 or less, 15000 or less, 10000 or less, 9000 or less, 8000 or less, 7000 or less, 6000 or less, 5000 or less, 4900 or less, 4800 or less, 4700 or less, 4600 or less, 4500 or less, 4400 or less, 4300 or less, 4200 or less, 4100 or less, 4000 or less, 3900 or less, 3700 or less, 3600 or less, 3500 or less, 3400 or less, 3300 or less, 3200 or less, 3090 or less, 3080 or less, 3030570 or less, 3060 or less, 3040 or less, 3030 or less, 3020 or less, 3000 or less, 2900 or less, 2700 or less, 2600 or less, 2500 or less, 2300 or less, 2000 or less, 2100 or less, 1800 or less, 1500, 1400 or less, or more, 2800 or less, or, 1200 or less, 1100 or less, or 1000 or less.

The lower limit of the relative expression amount of the PLIN2 gene with respect to the expression amount of the NEFM gene may be, for example, 250 or more, 260 or more, 270 or more, 280 or more, 290 or more, 300 or more, 310 or more, 320 or more, 330 or more, 340 or more, 350 or more, 360 or more, 370 or more, 380 or more, 390 or more, 400 or more, 410 or more, 420 or more, 430 or more, 440 or more, 450 or more, 460 or more, 470 or more, 480 or more, 490 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more.

The detection of each gene and/or the measurement of the expression level thereof can be carried out by, for example, microarray, RT-PCR, quantitative RT-PCR or Northern blot hybridization, but is not limited thereto.

As a method for measuring the relative expression amounts of the respective genes with respect to the expression amount of the SDHA gene and the expression amount of the PLIN2 gene with respect to the expression amount of the NEFM gene, a microarray can be used. Specifically, the microarray can be performed according to the following steps (1) to (5). The following steps (3) to (5) may be carried out by RIKEN GENESIS.

(1) The cryopreserved cell population was thawed and recovered by centrifugation. The recovered cell population was washed with Phosphate Buffered Saline (PBS), and the cells were recovered by centrifugation.

(2) Total RNA was extracted from the cell population and purified using an RNA extraction kit (RNeasy Plus Mini kit (QIAGEN)).

(3) Using the purified total RNA as a template, cDNA was synthesized by reverse transcription, and further transcribed from the synthesized cDNA to cRNA by in vitro transcription, and biotin labeling was performed.

(4) Biotin-labeled cRNA was added to the hybridization buffer, and hybridization was carried out for 16 hours on a Human Genegenome U133A 2.0.0 Array (manufactured by Affymetrix). Washing was performed with a GeneChip fluids Station 450 (manufactured by Affymetrix), after phycoerythrin staining, scanning was performed with a GeneChip Scanner 30007G (manufactured by Affymetrix), image analysis was performed with agcc (Affymetrix GeneChip Command conditioner software) (manufactured by Affymetrix), and digitization was performed with an Affymetrix Expression conditioner (manufactured by Affymetrix).

(5) The numerical data files were compared and analyzed using analysis software GeneSpring GX (Agilent Technologies, Inc.). The relative expression level of each gene in each cell population with respect to the expression level of the SDHA gene or NEFM gene was calculated.

Quantitative RT-PCR can be used as a method for measuring the relative expression level of each gene with respect to the expression level of SDHA gene and the relative expression level of PLIN2 gene with respect to the expression level of NEFM gene. Specifically, quantitative RT-PCR can be performed according to the following procedure A (comparative Ct method) or procedure B (calibration curve method).

(step A: comparative Ct method)

(1) The cryopreserved cell population was thawed and recovered by centrifugation. The recovered cell population was washed with Phosphate Buffered Saline (PBS) and recovered by centrifugation.

(2) Total RNA was extracted and purified from the cell population using an RNA extraction kit (RNeasy Plus Mini kit (QIAGEN)).

(3) cDNA was synthesized by reverse transcription reaction using the purified total RNA as a template and ReverTra Ace qPCR RT Master Mixwith gDNAremover (manufactured by Toyo Co., Ltd.).

(4) The PCR reaction was carried out using KOD SYBR qPCR Mix (manufactured by Toyo Boseki Co., Ltd.) with the synthesized cDNA as a template. The liquid volume of the reaction solution was 20. mu.L. The PCR conditions were 40 cycles (98 ℃ for 10 seconds, 60 ℃ for 10 seconds, and 68 ℃ for 30 seconds) using a StepOneNus Real-Time PCR System (manufactured by Thermo Fisher Scientific Co., Ltd.) maintained at 98 ℃ for 2 minutes.

(5) The relative expression amount (X) of the gene to be measured with respect to the gene to be compared is represented by X ═ 2^{-^(Y-Z)}And (4) calculating. Here, Y is a Ct value of a gene to be measured, and Z is a Ct value of a gene to be compared.

(step B: calibration Curve method)

(3) cDNA was synthesized by reverse transcription reaction using the purified total RNA as a template and ReverTra Ace qPCR RT Master Mix with DNA Remover (Toyo Co., Ltd.).

(4) The PCR reaction was carried out using KOD SYBR qPCR Mix (manufactured by Toyo Boseki Co., Ltd.) with the synthesized cDNA as a template. The liquid volume of the reaction solution was 20. mu.L. The PCR conditions were 40 cycles (98 ℃ for 10 seconds, 60 ℃ for 10 seconds, and 68 ℃ for 30 seconds) using a StepOneNus Real-Time PCR System (manufactured by Thermo Fisher Scientific Co., Ltd.) maintained at 98 ℃ for 2 minutes. As a sample for drawing a calibration curve, a sample obtained by diluting the synthesized cDNA in an equal-fold (double-fold) scale of 1 to 5 was used.

(5) The expression levels of the genes to be compared and the genes to be measured, which were indicated by StepOneNus Real-Time PCR System (Thermo Fisher Scientific Co., Ltd.), were recorded.

(6) The relative expression amount (W) of the gene of the measurement target to the comparison target was calculated as W ═ α/β. Here, α is the expression level of the gene to be measured, and β is the expression level of the gene to be compared.

In the above-mentioned quantitative RT-PCR, the following primer sequences can be used.

PLIN 2-f: 5-GCTGAGCACATTGAGTCACG-3 (SEQ ID NO. 7)

PLIN 2-r: 5-TGGTACACCTTGGATGTTGG-3 (SEQ ID NO. 8)

NEFM-f: 5-TAGAAATCGCTGCGTACAGAAAAC-3 (SEQ ID NO. 9)

NEFM-r: 5-TGCTTCCTGCAAATGTGCTAA-3 (SEQ ID NO. 10)

SDHA-f: 5-TGGGAACAAGAGGGCATCTG-3 (SEQ ID NO. 11)

SDHA-r: 5-CCACCACTGCATCAAATTCATG-3 (SEQ ID NO. 12)

The timing for measuring the gene expression level is not particularly limited, and examples thereof include: the present invention is not limited to the above-described examples, and the examples include the following examples, such as immediately after the isolation of cells from a biological sample, during the culture step, after purification in the culture step, immediately after n passages (n represents an integer of 1 or more), during the maintenance of culture, before cryopreservation, after thawing, or before the preparation becomes a pharmaceutical composition.

According to one embodiment of the present invention, the population of cells comprising adherent stem cells provided by the present invention can satisfy a ratio of adherent stem cells positive for CD105, CD73, and/or CD90 of 90% or more.

CD105 refers to the differentiated population 105 and is a protein known as Endoglin.

CD73 refers to clade 73 and is a protein known as a 5-nucleotidase or Ecto-5' -nucleotidase.

CD90 refers to the clade 90 and is a protein known as Thy-1.

The percentage of adherent stem cells that are positive for CD105 in the cell population may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

The percentage of adherent stem cells positive for CD73 in the cell population may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

The percentage of adherent stem cells positive for CD90 in the cell population may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

According to one embodiment of the present invention, the population of cells including adherent stem cells provided by the present invention can satisfy a requirement that the rate of adherent stem cells positive for CD166 is 30% or more.

CD166 refers to a differentiated population 166, a protein known as activated leukocyte adhesion molecule (ALCAM).

The percentage of adherent stem cells that are positive for CD166 in the cell population may be 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

According to one embodiment of the present invention, the population of adherent stem cells provided by the present invention may satisfy a ratio of adherent stem cells negative for CD45, CD34, CD11b, CD79alpha, CD19, and/or HLA-DR of 95% or more.

CD45 refers to the differentiation group 45 and is a protein known as PTPRC (protein tyrosine phosphatase, receptor type, C), or LCA (leukocyte common antigen).

CD34 refers to the differentiated population 34, a protein known as Hematographic promoter cell antigen CD 34.

CD11b refers to the differentiation group 11b and is a protein known as ITGAM (integrin, alpha M).

CD79alpha refers to the differentiation group 79alpha, and is a protein known as the alpha chain of B-cell antigen receptor complex-associated protein (B-cell antigen receptor complex-associated protein alpha chain) or MB-1 membrane glycoprotein (membrane glycoprotein).

CD19 refers to the differentiated population 19 and is a protein known as the B lymphocyte antigen CD19(B-lymphocyte antigen CD 19).

HLA-DR refers to Human leucocyte antigen-antigen D related, a protein known as MHC class II cell surface receptor.

The percentage of adherent stem cells that are negative for CD45 in the cell population may be 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

The percentage of adherent stem cells that are negative for CD34 in the cell population may be 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

The ratio of adherent stem cells that are negative for CD11b in the cell population may be 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

The percentage of adherent stem cells that are negative for CD79alpha in the population of cells may be 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

The percentage of adherent stem cells that are negative for CD19 in the cell population may be 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

The ratio of adherent stem cells in the cell population that are negative for HLA-DR may be 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

According to one embodiment of the present invention, the population of cells comprising adherent stem cells provided by the present invention can satisfy a ratio of adherent stem cells negative for CD324 and/or CD326 of 95% or more.

CD324 refers to the differentiation group 324 and is a protein known as Cadherin 1, or E-Cadherin, encoded by the CDH1 gene.

CD326 refers to a differentiation group 326, a protein known as Epithelial cell adhesion molecule (epitlial cell adhesion molecule) encoded by EPCAM gene.

The percentage of adherent stem cells that are negative for CD324 in the population of cells may be 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

The percentage of adherent stem cells in the population that are negative for CD326 may be 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

The measurement of each surface antigen can be performed by, for example, flow cytometry or cell staining, but is not limited thereto. In flow cytometry using a fluorescently labeled antibody, a cell is determined to be "positive" for the surface antigen when it fluoresces more strongly than a negative control (isotype control) is detected. Any antibody can be used as the fluorescently labeled antibody, and examples thereof include, but are not limited to, antibodies labeled with Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE), Allophycocyanin (APC), and the like. When a stained or fluorescent cell is observed under a microscope during cell staining, the cell is judged to be "positive" for the surface antigen. The cell staining may be immune cell staining using an antibody or non-immune cell staining without using an antibody.

Specifically, the ratio of cells positive to each surface antigen (positive rate) can be measured by the following steps (1) to (8) using dot-plot analysis by flow cytometry.

(2) After fixation of the cell population with 4% paraformaldehyde, washing with Phosphate Buffered Saline (PBS) and preparation of 1.0 × 10 with 2% BSA/PBS⁶Cell suspension per mL. The cell suspensions were dispensed in 100. mu.L each.

(3) The cell suspension after the distribution was centrifuged, and 100. mu.L of 0.5% BSA/PBS was added to each of the obtained cell pellets. Subsequently, antibodies corresponding to the respective surface antigens or antibodies for isotype control thereof are added. The reaction solutions were mixed and then allowed to stand at 4 ℃ for 20 minutes.

(4) 0.5% BSA/PBS was added, the cell population was washed by centrifugation, suspended in 0.5% BSA/PBS, and filtered through a cell filter (35 μm nylon mesh filter) (model 352235, Corning).

(5) Using BD Accuri^TMThe cell suspension obtained by filtration through the filter was analyzed by C6Flow Cytometer (Becton, Dickinson and Company) as ALL Event 10000.

(6) The fluorescence intensity of the antibody-labeled dye is measured on the vertical axis in SSC (side scattered light) (numerical range: 0 to 16777215) and on the horizontal axis in 10¹Above and 10^7.2Below) was plotted.

(7) In the dot diagram, the entire region (gate) in which the fluorescence intensity of all cells measured with the antibody for isotype control is higher than 1.0% was selected.

(8) The ratio of cells contained in the gate selected in (7) among all the cells measured with the antibody corresponding to each surface antigen was calculated.

The ratio of cells negative to each surface antigen (negative rate) was calculated by the following formula.

The percent of negative results (%) -100-positive results

The timing of detecting the surface antigen is not particularly limited, and examples thereof include: the present invention is not limited to the above-described examples, and the examples include the following examples, such as immediately after the isolation of cells from a biological sample, during the culture step, after purification in the culture step, immediately after n passages (n represents an integer of 1 or more), during the maintenance of culture, before cryopreservation, after thawing, or before the preparation becomes a pharmaceutical composition.

The proliferation ability of the cell population comprising adherent stem cells provided by the present invention can be evaluated by the specific proliferation rate. The specific proliferation rate is defined as the increase in the number of cells per unit time, and is expressed as μ ═ ln (mt2/mt1)/(t2-t 1). Here, t1 and t2 indicate the number of days of culture, mt1 indicates the number of cells on day t1, and mt2 indicates the number of cells on day t2 (where t2 > t 1). Therefore, the unit of the specific growth rate is (one/day) ═ 1/day.

From the viewpoint of obtaining the number of cells required for production of a pharmaceutical composition at low cost and in a short time, the specific growth rate of the cell population containing adherent stem cells of the present invention is preferably 0.40(1/day) or more, more preferably 0.41(1/day) or more, further preferably 0.42(1/day) or more, further preferably 0.43(1/day) or more, further preferably 0.44(1/day) or more, further preferably 0.45(1/day) or more, further preferably 0.46(1/day) or more, further preferably 0.48(1/day) or more, further preferably 0.50(1/day) or more, further preferably 0.52(1/day) or more, further preferably 0.54(1/day) or more, further preferably 0.55(1/day) or more, further preferably 0.56(1/day) or more, more preferably 0.58(1/day) or more, further preferably 0.60(1/day) or more, further preferably 0.62(1/day) or more, further preferably 0.64(1/day) or more, further preferably 0.66(1/day) or more, further preferably 0.68(1/day) or more, further preferably 0.70(1/day) or more, further preferably 0.72(1/day) or more, further preferably 0.74(1/day) or more, further preferably 0.76(1/day) or more, further preferably 0.78(1/day) or more, further preferably 0.80(1/day) or more, further preferably 0.82(1/day) or more, further preferably 0.84(1/day) or more, further preferably 0.86(1/day) or more, and particularly preferably 0.87(1/day) or more. The upper limit of the specific growth rate of the cell population including adherent stem cells according to the present invention is not particularly limited, and is, for example, 1.0(1/day) or less or 0.90(1/day) or less.

In the cell population of the present invention, the adherent stem cells may be cultured until after preferably 20 days, further preferably 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 105 days, or 110 days after the start of the in vitro culture.

The passable number of the cell population including adherent stem cells provided by the present invention is 1 or more, preferably 2 or more, more preferably 3 or more, further preferably 4 or more, further preferably 5 or more, further preferably 6 or more, further preferably 8 or more, further preferably 10 or more, further preferably 12 or more, further preferably 14 or more, further preferably 16 or more, further preferably 18 or more, further preferably 20 or more, further preferably 22 or more, further preferably 24 or more, and further preferably 25 or more. The upper limit of the passable number is not particularly limited, and is, for example, 50 times or less, 45 times or less, 40 times or less, 35 times or less, or 30 times or less.

The cell populations comprising adherent stem cells provided by the invention are optionally passaged. The lower limit of the number of passages is preferably 1 or more, more preferably 2 or more, further preferably 3 or more, further preferably 4 or more, and further preferably 5 or more. The upper limit of the number of passages is preferably 25 or less, more preferably 20 or less, further preferably 15 or less, and further preferably 10 or less.

The cell population containing adherent stem cells provided by the present invention can be population-multiplied preferably 10 times or more, more preferably 20 times or more, 30 times or more, 40 times or more, 50 times or more, or 60 times or more. The cell population including adherent stem cells provided by the present invention may be multiplied 100 times or less, 90 times or less, 80 times or less, or 70 times or less, for example, but is not limited thereto.

The cell populations comprising adherent stem cells provided by the invention are optionally population-multiplied. The lower limit of the number of population doublings is preferably 5 or more, more preferably 10 or more, further preferably 15 or more, further preferably 20 or more, further preferably 25 or more, further preferably 30 or more. The upper limit of the number of population doublings is preferably 60 or less, more preferably 55 or less, and still more preferably 50 or less.

Population doubling is the number of cell population divisions during a given culture period, by [ log ]₁₀(number of cells at the end of culture) -log₁₀(number of cells at the beginning of culture)]/log₁₀(2) The formula of (4) is calculated. Is proceeding toIn subculture, the number of population doublings per passage is calculated by the above formula, and then accumulated, thereby calculating the total number of population doublings.

The immunosuppressive effect of the cell population including adherent stem cells provided by the present invention can be evaluated by the expression level of TNFAIP6 (tumor necrosis factor, alpha-induced protein 6) gene, the proliferation inhibition rate of human peripheral blood mononuclear cells (hPBMC) in the mixed lymphocyte reaction test (MLR test), and/or an animal test.

The sequence of TNFAIP6 (alpha-induced protein 6) gene was registered as ID: 7130. TNFAIP6 is a gene having a base sequence represented by SEQ ID NO. 13 or a gene encoding a polypeptide having an amino acid sequence represented by SEQ ID NO. 14.

The expression level of TNFAIP6 gene can be evaluated by, for example, microarray, RT-PCR, quantitative RT-PCR or Northern blot hybridization, but is not limited thereto.

As a method for measuring the relative expression level of TNFAIP6 gene with respect to the expression level of SDHA gene, a microarray can be used. The microarray may be performed according to the steps described above.

The lower limit of the relative expression amount of TNFAIP6 gene with respect to the expression amount of the SDHA gene may be 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, 1.0 or more, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, or 2.0 or more. The upper limit of the relative expression amount of TNFAIP6 gene with respect to the expression amount of the SDHA gene is not particularly limited, and is, for example, 4.0 or less, 3.0 or less, or 2.0 or less.

The inhibition rate of human peripheral blood mononuclear cells (hPBMC) proliferation in the mixed lymphocyte reaction test (MLR test) can be evaluated according to the following procedures (1) to (6).

(1) The target cell population was designated 2 × 10⁴One cell/well was inoculated in a 96-well plate for adherent culture, and adherent culture was performed for 24 hours in α MEM containing 10% bovine fetal serum (FBS) (inactivated) in final concentration.

(2) Mitomycin C was added to the cell population after 24 hours of adherent culture at a final concentration of 10. mu.g/mL and incubated at 37 ℃ for 2 hours.

(3) As a co-culture group, 2 × 10 was inoculated into a cell population containing mitomycin C⁵Individual/well hBMC as a separate culture group (control group), wells containing no cell population were inoculated with 2 × 10⁵Individual/well hPBMC. Phytohemagglutinin (PHA) was added to each of the groups (co-culture group and single culture group) at a final concentration of 2.5. mu.g/mL, and the mixture was cultured at 37 ℃ for 96 hours.

(4) After 96 hours of culture, the supernatant containing the hBMC was collected, and the total cell count and the dead cell count of the hBMC were measured by an automatic cell counter (ChemOMETec Co., Ltd.).

(5) The number of viable cells of hPBMC was calculated by the following formula.

Number of viable cells of hPPMC-total number of dead cells of hPPMC

(6) The growth inhibition rate of hBMC was calculated by the following equation.

The proliferation inhibition ratio (%) of hPBMC was 100 — (viable cell number of hPBMC in co-culture group/viable cell number of hPBMC in single culture group) × 100

The timing of evaluating the above-mentioned immunosuppressive action is not particularly limited, and examples thereof include: the present invention is not limited to the above-described examples, and the examples include the following examples, such as immediately after the isolation of cells from a biological sample, during the culture step, after purification in the culture step, immediately after n passages (n represents an integer of 1 or more), during the maintenance of culture, before cryopreservation, after thawing, or before the preparation becomes a pharmaceutical composition.

The cell survival rate of the cell population including adherent stem cells provided by the present invention can be measured by, for example, trypan blue staining, MTT (3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyltetrazolium bromide) assay, and the like, but is not limited thereto.

The cell survival rate of the cell population including adherent stem cells provided by the present invention is preferably 70% or more, more preferably 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

The source of the adherent stem cells is not particularly limited, and, for example, adherent stem cells derived from fetal appendages, bone marrow, fat, or dental pulp can be used. The adherent stem cells are preferably adherent stem cells from a fetal appendage, more preferably adherent stem cells from an amniotic membrane. Adherent stem cells are adherent stem cells isolated from a sample of an autologous, allogeneic or xenogeneic organism, preferably adherent stem cells isolated from a sample of an allogeneic organism.

The adherent stem cells are adherent stem cells subjected to or not subjected to gene recombination, and preferably are adherent stem cells not subjected to gene recombination.

The cell population of the invention can comprise any number of adherent stem cells the cell population of the invention can comprise, for example, 1.0 × 10¹1.0 × 10²1.0 × 10³1.0 × 10⁴1.0 × 10⁵1.0 × 10⁶1.0 × 10⁷1.0 × 10⁸1.0 × 10⁹1.0 × 10¹⁰1.0 × 10¹¹1.0 × 10¹²1.0 × 10¹³More or less adherent stem cells, but not limited thereto.

The cell population of the present invention may contain an arbitrary component in addition to the adherent stem cells. Examples of the above-mentioned components include: salts, polysaccharides (e.g., hydroxyethyl starch (HES), dextran, etc.), proteins (e.g., albumin, etc.), dimethyl sulfoxide (DMSO), amino acids, medium components (e.g., components contained in RPMI1640 medium), etc., but the present invention is not limited thereto.

The cell population of the present invention may be stored in a frozen state until just before use. The cell population may contain a cryopreservation solution in addition to the adherent stem cells. As the cryopreservation solution, a commercially available cryopreservation solution can be used. Examples thereof include: CP-1 (registered trademark) (manufactured by Toyobo pharmaceutical industries Co., Ltd.), BAMBANKER (manufactured by Lymphotec Co., Ltd.), STEM-CELLBANKER (manufactured by Nippon Kagaku K.K.), Reprocryo RM (manufactured by REPROELL Co., Ltd.), CryoNovo (manufactured by Akron Biotechnology Co., Ltd.), MSC Freezing Solution (manufactured by biologicals industries Co., Ltd.), CryoStor (manufactured by HemaCare Co., Ltd.), and the like, but are not limited thereto.

The cell population of the present invention may be provided in the form of a composition combined with a medium. As the medium, a liquid medium (for example, a culture medium, dimethyl sulfoxide (DMSO), a cryopreservation solution, or a pharmaceutically acceptable vehicle described later) can be preferably used.

The cell concentration of the composition comprising the cell population of the invention and the mediator may be, for example, 1.0 × 10¹1.0 × 10/mL²1.0 × 10/mL³1.0 × 10/mL⁴1.0 × 10/mL⁵1.0 × 10/mL⁶1.0 × 10/mL⁷1.0 × 10/mL⁸1.0 × 10/mL⁹1.0 × 10/mL¹⁰The cell concentration is not less than or equal to one/mL, but is not limited thereto.

[3] Method for producing cell population comprising adherent stem cells

The method for producing a cell population comprising adherent stem cells of the present invention comprises: a cell population having the cell characteristics of (a) and (b) shown below was obtained.

(a) Positive expression of the PLIN2 gene and NEFM gene in the cell population, and (b) a relative expression level of the PLIN2 gene in the cell population with respect to the expression level of the SDHA gene of 1.50 or more.

That is, the method for producing a cell population including adherent stem cells of the present invention comprises: preparing a cell population comprising adherent stem cells under conditions that maintain the cell characteristics of (a) and (b). The conditions of (a) and (b) are indices for forming a cell population including adherent stem cells exhibiting high immunosuppressive activity and/or proliferation property, and the production method of the present invention is not particularly limited as long as the indices are satisfied.

The manufacturing method of the present invention may include: a cell population obtaining step of obtaining a cell population including adherent stem cells by subjecting a biological sample (e.g., a fetal appendage such as an amniotic membrane) including the adherent stem cells to an enzyme treatment. The above-mentioned cell population obtaining step may include a step of obtaining an amnion by a caesarean section. The cell population obtaining step may include a step of washing a biological sample containing adherent stem cells.

The amniotic membrane is composed of an epithelial cell layer and an extracellular matrix layer, the latter comprising amnion adherent stem cells. The amniotic epithelial cells are characterized by expressing epithelial adhesion factor (EpCAM: CD326) as in other epithelial cells, whereas the amniotic adherent stem cells do not express the epithelial-specific surface antigen marker for CD326 and can be readily differentiated by flow cytometry.

The cell population comprising adherent stem cells of the invention is preferably a cell population obtained by treating a sample comprising an epithelial cell layer and an extracellular matrix layer collected from a fetal appendage with at least collagenase.

The enzymatic treatment of the sample collected from the fetal appendage, preferably a sample comprising an epithelial cell layer and an extracellular matrix layer, is preferably a treatment with an enzyme (or a combination thereof) that is capable of freeing adherent stem cells comprised in the extracellular matrix layer of the fetal appendage and that does not break down the epithelial cell layer. The enzyme is not particularly limited, and examples thereof include collagenase and/or metalloprotease. Examples of the metalloprotease include thermolysin and/or Dispase (Dispase) which are metalloproteases cleaving the N-terminal side of the nonpolar amino acid, and are not particularly limited.

The collagenase activity concentration is preferably 50PU/ml or more, more preferably 100PU/ml or more, and still more preferably 200PU/ml or more. The collagenase activity concentration is not particularly limited, but is, for example, 1000PU/ml or less, 900PU/ml or less, 800PU/ml or less, 700PU/ml or less, 600PU/ml or less, or 500PU/ml or less. Here, PU (protease Unit) is defined as the amount of enzyme that decomposes 1ug of FITC-collagen at pH7.5 at 30 ℃ for 1 minute.

The activity concentration of the metalloprotease (e.g., thermolysin and/or Dispase) is preferably 50PU/ml or more, more preferably 100PU/ml or more, and still more preferably 200PU/ml or more. The active concentration of the metalloprotease is preferably 1000PU/ml or less, more preferably 900PU/ml or less, further preferably 800PU/ml or less, further preferably 700PU/ml or less, further preferably 600PU/ml or less, further preferably 500PU/ml or less. Here, in the embodiment using Dispase (Dispase) as the metalloprotease, PU (protease Unit) is defined as the amount of enzyme that releases 1ug of tyrosine-equivalent amino acid from casein (casein lactate) at pH7.5 at 30 ℃ for 1 minute. Within the above-mentioned range of enzyme concentration, it is possible to prevent the contamination of epithelial cells contained in the epithelial cell layer of the fetal appendage and to efficiently release adherent stem cells contained in the extracellular matrix layer. The preferred combination of concentrations of collagenase and/or metalloprotease can be determined by microscopic observation of the enzyme-treated fetal appendages, flow cytometry of the cells obtained.

From the viewpoint of efficiently recovering living cells, it is preferable to treat the fetal appendages with a combination of collagenase and metalloprotease at the same time. In this case, thermolysin and/or Dispase (Dispase) can be used as the metalloprotease, but the metalloprotease is not limited thereto. Adherent stem cells can be obtained simply by treating a fetal appendage with an enzyme solution containing collagenase and metalloprotease only once. In addition, the risk of contamination with bacteria, viruses, and the like can be reduced by performing the treatment at the same time.

The enzymatic treatment of fetal appendages is preferably carried out by immersing amnion washed with a washing solution such as physiological saline or Hank's balanced salt solution in an enzymatic solution and stirring the amnion with a stirring device. As the stirring device, for example, a stirrer or a vibrator can be used from the viewpoint of efficiently releasing the adherent stem cells contained in the extracellular matrix layer of the fetal appendage, but the stirring device is not limited thereto. The stirring speed is not particularly limited, and when a stirrer or a vibrator is used, it is, for example, 10rpm or more, 30rpm or more, or 50rpm or more. The stirring speed is not particularly limited, and when a stirrer or a vibrator is used, it is, for example, 100rpm or less, 80rpm or less, or 60rpm or less. The enzyme treatment time is not particularly limited, and is, for example, 10 minutes or more, 30 minutes or more, 50 minutes or more, 70 minutes or more, or 90 minutes or more. The enzyme treatment time is not particularly limited, and is, for example, 6 hours or less, 4 hours or less, 2 hours or less, and 100 minutes or less. The enzyme treatment temperature is not particularly limited, and is, for example, 16 ℃ or higher, 20 ℃ or higher, 24 ℃ or higher, 28 ℃ or higher, 32 ℃ or higher, or 36 ℃ or higher. The enzyme treatment temperature is not particularly limited, and is, for example, 40 ℃ or lower, 39 ℃ or lower, or 38 ℃ or lower.

In the production method of the present invention, the free adherent stem cells can be separated and/or recovered from the enzyme solution containing the free adherent stem cells by a known method such as a filter, centrifugation, a hollow fiber separation membrane, or a cell sorter, as desired. The enzyme solution comprising free adherent stem cells is preferably filtered using a filter. In the method of filtering the enzyme solution with a filter, only the free cells are passed through the filter, and the undecomposed epithelial cell layer remains on the filter without passing through the filter, so that not only can the free adherent stem cells be easily separated and/or recovered, but also the risk of contamination with bacteria, viruses, and the like can be reduced. The filter is not particularly limited, and examples thereof include a mesh filter. The pore diameter (size of mesh) of the mesh filter is not particularly limited, and is, for example, 40 μm or more, 60 μm or more, 80 μm or more, or 90 μm or more. The pore size of the mesh filter is not particularly limited, but is, for example, 200 μm or less, 180 μm or less, 160 μm or less, 140 μm or less, 120 μm or less, or 100 μm or less. The filtration rate is not particularly limited, and when the pore diameter of the mesh filter is set to the above range, the enzyme solution containing adherent stem cells can be filtered by allowing it to naturally fall, thereby preventing the decrease in cell survival rate.

As a material of the mesh filter, nylon is preferably used. Tubes having a nylon mesh filter of 40 μm, 70 μm, 95 μm or 100 μm, such as Falcon cell filters, which are widely used for research purposes, can be used. Medical mesh cloths (nylon and polyester) used for hemodialysis and the like can be used. Further, an arterial filter (polyester mesh filter, pore size: 40 μm to 120 μm) used in extracorporeal circulation may be used. Other materials, such as stainless steel mesh filters, etc., may also be used.

When the adherent stem cells are passed through the filter, they are preferably allowed to drip (fall freely). A forced passage of a filter such as suction using a pump or the like may be used, and a pressure as low as possible is preferable in order to avoid damage to cells.

Adherent stem cells passed through the filter can be recovered by centrifugation after diluting the filtrate with a double or more amount of the medium or the balanced salt buffer. Examples of the balanced salt buffer include, but are not limited to, physiological saline, Dulbecco's Phosphate Buffer (DPBS), Earle's Balanced Salt Solution (EBSS), Hank's Balanced Salt Solution (HBSS), and Phosphate Buffer Solution (PBS).

The cell population obtained in the cell population obtaining step is prepared under the conditions that (a) the expression of the PLIN2 gene and NEFM gene of the cell population is positive, and (b) the relative expression level of the PLIN2 gene of the cell population with respect to the expression level of the SDHA gene is 1.50 or more. The above conditions are useful as an index for obtaining a cell population containing adherent stem cells exhibiting high immunosuppressive action and/or proliferation. The production method is not particularly limited as long as the above-mentioned index is satisfied. Examples of such a method include a method in which a cell group satisfying the above (a) is sorted out by a cell sorter, and then a cell group satisfying the above (b) is selected from the obtained cell groups; selecting a cell population satisfying the above (b), and then sorting the obtained cell population by a cell sorter. Further, another production method satisfying the above-mentioned criteria includes culturing a cell population under conditions satisfying the above-mentioned (a) and (b).

The culture method satisfying the above-mentioned criteria includes, for example, repeating the culture at 100 to 20000 cells/cm²Density of cellsAnd (3) inoculating the population into an uncoated plastic culture vessel and culturing the population. The lower limit of the density at the time of seeding of the cell population is more preferably 200 cells/cm²Above, more preferably 400 pieces/cm²More preferably 600 pieces/cm²Above, more preferably 800 pieces/cm²More preferably 1000 pieces/cm²More preferably 1200 pieces/cm²The number of the molecules is more preferably 1400/cm²More preferably 1600/cm²Above, more preferably 1800 pieces/cm²More preferably 2000 pieces/cm²The above. The upper limit of the density at the time of seeding of the cell population is more preferably 18000 cells/cm²More preferably 16000 pieces/cm²More preferably 14000 cells/cm²Hereinafter, more preferably 12000 pieces/cm²Hereinafter, 10000 pieces/cm are more preferable²Hereinafter, more preferably 8000 pieces/cm²The following.

As another culturing method satisfying the above-mentioned criteria, for example, a method of repeating the culturing at a rate of 100 to 20000 cells/cm²The step of culturing the cells by inoculating the cell population in a culture vessel made of plastic coated with a coating agent. The preferred conditions for the density at the time of seeding the cell population are the same as those described above.

Examples of the coating agent include: extracellular matrix, fibronectin, vitronectin, osteopontin, laminin, entactin (entactin), collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, gelatin, poly-L-ornithine, poly-D-lysine, Matrigel (registered trademark) matrix, but is not limited thereto.

The medium used for the above culture can be prepared by using an arbitrary liquid medium for animal cell culture as a basal medium and adding other components (albumin, serum-replacement reagent, growth factor, human platelet lysate, etc.) as needed.

As the basal Medium, there may be used, but not particularly limited to, BME Medium, BGJb Medium, CMRL1066 Medium, GlasgowMEM Medium, Improved MEM stretch Medium, IMDM Medium (Iscove's Modified Dulbecco's Medium), Medium 199 Medium, Eagle MEM Medium, alpha MEM (alpha MEM) Medium, DMEM Medium (Dulbecco's Modified Eagle's Medium), Ham's F10 Medium, Ham's F12 Medium, RPMI1640 Medium, Fischer's Medium, and mixed Medium thereof (for example, DMEM/F12 Medium (Dulbecco's Modified Eagle's Nuxture F-12 m)).

The medium used for the above culture may be a commercially available serum-free medium. Examples thereof include: STK1, STK2(DS PHARMA BIOMEDICAL Co., Ltd.), EXPREP MSC Medium (BioMimetics Symphies Co., Ltd.), Corning stemgro human adherent stem cell culture Medium (Corning Co., Ltd.), and the like, but are not particularly limited.

Examples of other components to be added to the basic medium include: albumin, serum replacement agents, growth factors, or human platelet lysate, and the like. In the above-described mode of adding albumin to the basal medium, the concentration of albumin is preferably more than 0.05% and 5% or less. In the above embodiment in which serum is added to the basal medium, the serum concentration is preferably 5% or more. In the case of the mode of adding the growth factor, a reagent (protein such as heparin, gel, polysaccharide, or the like) for stabilizing the growth factor in the culture medium may be further added in addition to the growth factor, or the stabilized growth factor may be previously added to the basic culture medium. Examples of the growth factor include, but are not limited to, Fibroblast Growth Factor (FGF), epithelial cell growth factor (EGF), Transforming Growth Factor (TGF), Vascular Endothelial Growth Factor (VEGF), platelet-derived growth factor (PDGF), and families thereof.

As another culture method satisfying the above-mentioned criteria, for example, a culture is carried out by adding human platelet lysate (hPL) to a basal medium used for the culture. The human platelet lysate is preferably subjected to inactivation of bacteria, viruses and/or sterilization treatment. As the human platelet lysate, a commercially available human platelet lysate can be used. Examples thereof include: stemulate (Cook Regentec), PLTMax (Mill Creek Life Science), UltraGRO (Aventacell BioMedial), PLUS (Compass Biomedical), etc., but the present invention is not limited thereto.

The final concentration of the human platelet lysate in the culture medium is preferably 1% or more, more preferably 2% or more, further preferably 3% or more, further preferably 4% or more, and further preferably 5% or more. The final concentration of the platelet lysate in the culture medium is preferably 20% or less, more preferably 18% or less, more preferably 16% or less, more preferably 14% or less, more preferably 12% or less, more preferably 10% or less, more preferably 9% or less, more preferably 8% or less, more preferably 7% or less, and more preferably 6% or less.

The timing of adding the human platelet lysate is not particularly limited, and examples thereof include: the culture medium may be used in the early stage of the culture step, during the culture step, after purification in the culture step, immediately after n passages (where n represents an integer of 1 or more), during the maintenance culture, before cryopreservation, after thawing, or the like.

The culture of adherent stem cells can be performed, for example, in the following steps. First, the cell suspension is centrifuged, the supernatant is removed, and the obtained cell pellet is suspended in a medium. Then, the cells were inoculated into a plastic culture vessel and the amount of CO was 3% to 5%₂The concentration, 37 ℃ environment, using culture medium culture, so that the confluency is 95% or less, as the medium, for example, α MEM, M199, or based on them medium, but not limited to, this, through the culture of the cells obtained is 1 after the culture.

The culture time of the above-mentioned 1 culture may be, for example, 2 to 15 days, and more specifically, 2 days, 3 days, 4 days, 5 days, 6 days, 8 days, 10 days, 12 days, 14 days, or 15 days.

The cells after the above-mentioned 1-time culture can be, for example, culturedThe cells were further subcultured and cultured as follows. First, the cells after 1 culture were treated by a cell detaching method and detached from the plastic culture container. Subsequently, the resulting cell suspension was centrifuged to remove the supernatant, and the resulting cell pellet was suspended in a medium. Finally, the cells were inoculated into a plastic culture vessel containing 3% to 5% CO₂The culture medium is used at a concentration of 95 ℃ or less, and the degree of confluency is 95% or less, and examples of the medium include α MEM, M199, and a medium based on these media, but the medium is not limited thereto, and the cells obtained by the above-mentioned passaging and culturing are cells after 1 passage, and the cells after n passages (n represents an integer of 1 or more) can be obtained by performing the same passaging and culturing.

As the cell separation method, for example, a cell separation agent can be used. Examples of the cell-releasing agent include trypsin, collagenase, Dispase (Dispase), ethylenediaminetetraacetic acid (EDTA), and the like, and are not particularly limited. As the cell-releasing agent, a commercially available cell-releasing agent can be used. Examples thereof include: examples of the trypsin-EDTA solution include, but are not limited to, trypsin-EDTA solution (manufactured by Thermo Fisher Scientific Co., Ltd.), TrypLE Select (manufactured by Thermo Fisher Scientific Co., Ltd.), Accutase (manufactured by Stemcell Technologies Co., Ltd.), and Accumax (manufactured by Stemcell Technologies Co., Ltd.). As the cell separation method, a physical cell separation method may be used, and for example, a cell scraper (manufactured by Corning) may be used. The cell separation method may be used alone or in combination of two or more.

According to the production method of the present invention, a cell population containing adherent stem cells exhibiting high immunosuppressive activity and/or proliferation can be obtained. Each 1 culture batchThe lower limit of the number of cells obtained in the next step (the number of cells obtained per unit surface area and per unit number of culture days) varies depending on the number of cells to be seeded, the seeding density, and the like, and is, for example, 5.0 × 10³(pieces/cm)²Day) above 6.0 × 10³(pieces/cm)²/day) above 8.0 × 10³(pieces/cm)²/day) above 1.0 × 10⁴(pieces/cm)²/day) above 1.1 × 10⁴(pieces/cm)²/day) or more, or 1.2 × 10⁴(pieces/cm)²Day) or more, and the upper limit of the number of cells obtained per 1 culture batch is not particularly limited, and is, for example, 1.0 × 10⁵(pieces/cm)²Day) below 8.0 × 10⁴(pieces/cm)²Day) of less than 6.0 × 10⁴(pieces/cm)²Day) of less than 4.0 × 10⁴(pieces/cm)²/day) or less, or 2.0 × 10⁴(pieces/cm)²And/day) below.

According to the production method of the present invention, a cell population containing adherent stem cells exhibiting high immunosuppressive activity and/or proliferation can be obtained. Thus, the adherent stem cells obtained by the production method of the present invention can be cultured preferably up to 20 days or later, more preferably up to 30 days or later, 40 days or later, 50 days or later, 60 days or later, 70 days or later, 80 days or later, 90 days or later, 100 days or later, or 110 days or later, after the start of in vitro culture.

The cell population containing adherent stem cells obtained by the production method of the present invention can be cultured until the population doubling time is preferably 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 60 or more after the in vitro culture is started.

The manufacturing method of the present invention may include: and (b) a step of identifying a cell population containing adherent stem cells exhibiting a high immunosuppressive action and/or a high proliferative property, using as an indicator a condition that (a) the expression of the PLIN2 gene and the NEFM gene of the cell population is positive and (b) the relative expression level of the PLIN2 gene of the cell population with respect to the expression level of the SDHA gene is 1.50 or more.

The method for identifying a cell population comprising the aforementioned adherent stem cells is preferably microarray and/or quantitative RT-PCR.

The relative expression amounts of the PLIN2 gene and/or NEFM gene with respect to the expression amount of the SDHA gene can be measured using a microarray according to the above-described procedure.

The timing of the above recognition is not particularly limited, and examples thereof include: the method for producing a pharmaceutical composition comprises the steps of isolating cells from a biological sample, culturing, purifying in the culturing step, maintaining the culture for n passages (n represents an integer of 1 or more), freezing before preservation, thawing after the culture, or preparing a pharmaceutical composition.

The manufacturing method of the present invention may further include: after identifying a cell population including the adherent stem cells using the (a) and (b) as indices, the identified cell population is selectively isolated. The method for selectively separating the identified cell population is not particularly limited, and examples thereof include: sorting of cell groups by a cell sorter, purification of cell groups by culture, and the like.

In addition, the manufacturing method of the present invention may include: and (d) performing cryopreservation of the cell population containing the adherent stem cells. In the embodiment including the step of cryopreserving the cell population, the cell population may be thawed and then, if necessary, identified, separated, collected and/or cultured. Alternatively, the cell population may be used as it is after thawing.

The method for cryopreservation of a cell population containing the aforementioned adherent stem cells is not particularly limited, and examples thereof include: program freezers, deep freezers, immersion in liquid nitrogen, and the like. When a program freezer is used, the temperature during freezing is preferably-30 ℃ or lower, -40 ℃ or lower, -50 ℃ or lower, -80 ℃ or lower, -90 ℃ or lower, -100 ℃ or lower, -150 ℃ or lower, -180 ℃ or lower, or-196 ℃ (liquid nitrogen temperature) or lower. When a program freezer is used, the freezing rate is preferably-1 ℃/min, -2 ℃/min, -5 ℃/min, -9 ℃/min, -10 ℃/min, -11 ℃/min, or-15 ℃/min. In the case of using a program freezer as the freezing method, the temperature can be stably lowered to a temperature between-50 ℃ and-30 ℃ (for example, -40 ℃) at a freezing rate of-2 ℃/min or more and-1 ℃/min or less, and further lowered to a temperature between-100 ℃ and-80 ℃ (for example, -90 ℃) at a freezing rate of-11 ℃/min or more and-9 ℃/min or less (for example, -10 ℃/min). In the case of using the above freezing method by immersing in liquid nitrogen, for example, the temperature may be rapidly lowered to-196 ℃ to freeze the sample, and then the sample may be cryopreserved in liquid nitrogen (gas phase).

When the freezing method is used for freezing, the cell population may be frozen in a state of being placed in an arbitrary storage container. Examples of the storage container include: a vial for cryopreservation, a bag for freezing, an infusion bag, etc., but the present invention is not limited thereto.

When the freezing method is employed, the cell population may be frozen in an arbitrary cryopreservation solution. As the cryopreservation solution, a commercially available cryopreservation solution can be used. Examples thereof include: CP-1 (registered trademark) (manufactured by Toyobo pharmaceutical Industries Co., Ltd.), BAMBANKER (manufactured by Lymphotec Co., Ltd.), STEM-CELLBANKER (manufactured by Nippon Kagaku K.K.), Reprocryo RM (manufactured by REPROELL Co., Ltd.), CryoNovo (manufactured by Akron Biotechnology Co., Ltd.), MSCFrezing Solution (manufactured by Biological Industries Co., Ltd.), CryoStor (manufactured by HemaCare Co., Ltd.), and the like, but is not limited thereto.

The cryopreservation solution may contain a polysaccharide at a predetermined concentration. The preferable concentration of the polysaccharide is, for example, 1 mass% or more, 2 mass% or more, 4 mass% or more, or 6 mass% or more. The preferable concentration of the polysaccharide is, for example, 20 mass% or less, 18 mass% or less, 16 mass% or less, 14 mass% or less, or 13 mass% or less. Examples of the polysaccharide include, but are not limited to, hydroxyethyl starch (HES) and Dextran (Dextran 40).

The cryopreservation solution may contain dimethyl sulfoxide (DMSO) at a given concentration. The preferable concentration of DMSO is, for example, 1 mass% or more, 2 mass% or more, 3 mass% or more, 4 mass% or more, or 5 mass% or more. Further, the preferable concentration of DMSO is, for example, 20 mass% or less, 18 mass% or less, 16 mass% or less, 14 mass% or less, 12 mass% or less, or 10 mass% or less.

The cryopreservation solution may contain albumin at a given concentration of more than 0 mass%. The preferable concentration of albumin is, for example, 1 mass% or more, 2 mass% or more, 3 mass% or more, or 4 mass% or more. The preferable concentration of albumin is, for example, 30 mass% or less, 20 mass% or less, 10 mass% or less, or 9 mass% or less. Examples of albumin include: bovine Serum Albumin (BSA), mouse albumin, human albumin, and the like, but is not limited thereto.

The manufacturing method of the present invention may include: and washing the cell population containing the adherent stem cells. Examples of the cleaning solution used in the step of cleaning the cell population containing the adherent stem cells include: physiological saline, Dulbecco's Phosphate Buffered Saline (DPBS), Earle's Balanced Salt Solution (EBSS), Hank's Balanced Salt Solution (HBSS), Phosphate Buffered Saline (PBS), and the like, but is not limited thereto. By washing the cell population, allergens, endotoxins, and the like can be reduced or removed. Examples of the above-mentioned allergens include: bovine Serum Albumin (BSA), porcine trypsin, porcine heparin, and the like, but is not limited thereto.

The manufacturing method of the present invention may include: and (d) removing, if necessary, undesired cell aggregates from the cell population containing the adherent stem cells. The step of removing undesired cell aggregates from a cell population comprising the aforementioned adherent stem cells may comprise: a step of filtering a cell population (cell suspension) containing adherent stem cells with a filter.

The manufacturing method of the present invention may include: and filling a storage container with a cell population containing the adherent stem cells. Examples of the storage container include: a vial for cryopreservation, a bag for freezing, an infusion bag, etc., but the present invention is not limited thereto.

[4] Method for monitoring immunosuppressive action and/or proliferation of adherent stem cells, method for evaluating donor and/or biological sample collected from donor, and method for judging and/or predicting optimal enzyme treatment conditions

In the present invention, the immunosuppressive effect and/or proliferation of adherent stem cells can be monitored by measuring (preferably by measuring over time) a cell population containing adherent stem cells under the condition that (a) the expression of the PLIN2 gene and NEFM gene of the cell population is positive and (b) the relative expression level of the PLIN2 gene of the cell population with respect to the expression level of the SDHA gene is 1.50 or more as an index. Examples of the steps necessary for the monitoring include a step of performing culture, a step of performing cryopreservation, and/or a step of performing a preparation.

By measuring the index over time in the step of culturing, the immunosuppressive action and/or the change in the proliferation of the adherent stem cells can be grasped quickly and easily and predicted. It is known that, in a cell population containing adherent stem cells satisfying the above-mentioned criterion, while the adherent stem cells maintain high immunosuppressive activity and/or proliferation, when the culture state continues for a value exceeding the above-mentioned criterion, it is predicted that the immunosuppressive activity and/or proliferation of the adherent stem cells are decreasing. When a decrease in immunosuppressive action and/or proliferation is read based on the index, the decrease in immunosuppressive action and/or proliferation of adherent stem cells can be suppressed by appropriately changing the culture conditions (such as the inoculation density, the change of the medium, the growth factor, and the serum) as needed. When the above-mentioned index is not satisfied, the cell population including adherent stem cells satisfying the above-mentioned index can be sorted by, for example, a cell sorting technique, and the immunosuppressive action and/or the decrease in the proliferation of the adherent stem cells can be suppressed by reseeding and subculturing the adherent stem cells in the cell population. In the initial stage of the culture, the culture conditions (such as the seed density, the medium, the growth factor, and the serum) may be designed so as to satisfy the above-mentioned index at the final stage of the step, and the above-mentioned index may be satisfied at least at the final stage.

In the present invention, the quality of the donor itself and/or a biological sample collected from the donor can be evaluated by obtaining a cell population containing adherent stem cells from the donor, measuring the presence or absence of the expression of the PLIN2 gene and NEFM gene, and the relative expression level of the PLIN2 gene with respect to the expression level of the SDHA gene, and measuring the relative expression level of the PLIN2 gene and NEFM gene, as positive indicators, and (b) the relative expression level of the PLIN2 gene with respect to the expression level of the SDHA gene of the above cell population is 1.50 or more. In the case where a cell population including adherent stem cells satisfying the above-described index is obtained (preferably, easily obtained), it can be confirmed that the quality of the donor and/or the biological sample collected from the donor is good from the viewpoint of having high immunosuppressive ability and/or high proliferative ability. On the other hand, when the relative expression level of the cell population including the adherent stem cells exceeds the index, the quality of the biological sample collected from the donor is poor, and thus, the immunosuppressive action and/or the decrease in the proliferation of the adherent stem cells can be suppressed by appropriately changing the culture conditions (such as the inoculation density, the change of the culture medium, the growth factor, and the serum). When the relative expression level of the cell population containing adherent stem cells exceeds the index, the cell population containing adherent stem cells satisfying the index is sorted by, for example, a cell sorting technique, and the adherent stem cells in the cell population are seeded and cultured, whereby the immunosuppressive action and/or the decrease in the proliferation of the adherent stem cells can be suppressed. In the initial stage of the culture, the culture conditions (such as the seed density, the medium, the growth factor, and the serum) may be designed so as to satisfy the above-mentioned index at the final stage of the step, and the above-mentioned index may be satisfied at least at the final stage. In the case of confirming the quality of the biological sample collected from the donor, the method for preparing and treating the biological sample and the method for culturing the cell population are not particularly limited, and any method may be employed.

In the present invention, the presence or absence of the expression of the PLIN2 gene and NEFM gene and the relative expression level of the PLIN2 gene with respect to the expression level of the SDHA gene are measured in a cell population obtained by subjecting a biological sample collected from a donor to an enzyme treatment, and the presence or absence of the expression level of the PLIN2 gene and NEFM gene, and (b) the relative expression level of the PLIN2 gene with respect to the expression level of the SDHA gene is 1.50 or more are evaluated as indicators, whereby the optimal enzyme treatment condition of the biological sample can be judged and/or predicted. In the case where a cell population including adherent stem cells satisfying the above-described index is obtained (preferably, easily obtained), it is judged and/or predicted that an enzyme treatment method of a biological sample collected from a donor is appropriate. On the other hand, if the culture state whose value exceeds the above-mentioned index continues, it is judged and/or predicted that the method of treating the biological sample collected from the donor with the enzyme is not appropriate. In the case of determining and/or predicting the optimal enzyme treatment method, the method for preparing and treating a biological sample and the method for culturing a cell population are not particularly limited, and any method can be used.

The above-mentioned index is not particularly limited as long as it is measured at a desired timing, and includes: the method for producing a pharmaceutical composition comprises the steps of isolating cells from a biological sample, culturing, purifying in the culturing step, maintaining the culture for n passages (n represents an integer of 1 or more), freezing before preservation, thawing after the culture, or preparing a pharmaceutical composition.

[5] Pharmaceutical composition

The cell population comprising adherent stem cells of the present invention can be used as a pharmaceutical composition. That is, according to the present invention, a pharmaceutical composition comprising the cell population of the present invention, and a pharmaceutically acceptable vehicle can be provided. In addition, according to the present invention, a pharmaceutical composition comprising the cell population containing adherent stem cells of the present invention and other cells that can be administered can be provided.

The pharmaceutical composition of the present invention can be used as a cell therapeutic agent, for example, a therapeutic agent for intractable diseases.

The pharmaceutical composition of the present invention can be used as a therapeutic agent for immune-related diseases. The above immune-related diseases can be treated by administering the pharmaceutical composition of the present invention to the treatment site in an amount capable of measuring the effect.

According to the present invention, a cell population comprising adherent stem cells of the present invention for use in a pharmaceutical composition can be provided.

According to the present invention, a cell population comprising adherent stem cells of the present invention for use in a cell therapeutic agent can be provided.

According to the present invention, a cell population comprising adherent stem cells of the invention for use in the treatment of an immune-related disease may be provided.

According to the present invention, there may be provided a cell population comprising adherent stem cells of the invention for administration to a patient or subject to suppress an immune response.

According to the present invention, there may be provided a method of transplanting cells to a patient or subject, and a method of treating a disease in a patient or subject, the method comprising: a step of administering to a patient or subject a therapeutically effective amount of a cell population comprising adherent stem cells of the invention.

According to the present invention, there may be provided the use of a cell population comprising adherent stem cells of the invention in the manufacture of a pharmaceutical composition.

According to the present invention, there may be provided the use of a cell population comprising adherent stem cells of the invention in the manufacture of a cell therapeutic.

According to the present invention, there may be provided use of a cell population comprising adherent stem cells of the invention in the manufacture of a therapeutic agent for an immune-related disease.

According to the present invention, there may be provided the use of a cell population comprising adherent stem cells of the invention in the manufacture of a therapeutic agent for administration to a patient or subject in order to suppress an immune response.

The pharmaceutical composition of the present invention can be prepared by diluting a cell population comprising adherent stem cells with a pharmaceutically acceptable vehicle. The pharmaceutically acceptable vehicle is not particularly limited as long as it is a solution that can be administered to a patient or a subject. The pharmaceutically acceptable vehicle may be an infusion formulation, and may be exemplified by: examples of the "solution" include water for injection, physiological saline, 5% glucose solution, ringer's solution, lactated ringer's solution, acetate ringer's solution, bicarbonate ringer's solution, amino acid solution, starting solution (solution 1), dehydration supplement solution (solution 2), maintenance infusion solution (solution 3), postoperative recovery solution (solution 4), and Plasma-Lyte a (registered trademark).

The "patient or subject" in the present specification is typically a human, but may be other animals. Examples of other animals include: mammals such as dogs, cats, cows, horses, pigs, goats, sheep, monkeys (cynomolgus monkey, macaque, common marmoset, japanese monkey), ferrets, rabbits, rodents (mice, rats, gerbils, guinea pigs, hamsters), and birds such as chickens and quails, but the present invention is not limited thereto.

The "immune-related disease" in the present specification is not particularly limited as long as it is a disease related to an immune response of a patient or a subject, and examples thereof include: graft Versus Host Disease (GVHD), Inflammatory Bowel Disease (IBD), Crohn's disease, ulcerative colitis, radiation enteritis, diabetes, systemic lupus erythematosus, connective tissue disease, bacteroidal polyposis, multiple sclerosis, psoriasis, autoimmune bullous disease, rheumatoid arthritis, etc.

The term "treatment" as used herein includes, but is not limited to, a significant improvement in at least one of the life prognosis, the functional prognosis, the survival rate, the weight loss, anemia, diarrhea, dark stool, abdominal pain, fever, decreased appetite, malnutrition, vomiting, fatigue, eruption, inflammation, ulcer, erosion, fistula, stenosis, ileus, internal bleeding, rectal bleeding, spasm, pain, decreased liver function, and blood test items of a patient or a subject.

The pharmaceutical compositions of the present invention may comprise any ingredient used in the treatment of a patient or subject. Examples of the above-mentioned components include: salts (e.g., physiological saline, ringer's solution, BICANATE infusion), polysaccharides (e.g., hydroxyethyl starch (HES), dextran, etc.), proteins (e.g., albumin, etc.), dimethyl sulfoxide (DMSO), amino acids, medium components (e.g., components contained in RPMI1640 medium), etc., but are not limited thereto.

The pharmaceutical composition of the present invention may contain various additives for increasing storage stability, isotonicity, absorbability and/or viscosity, for example, emulsifiers, dispersants, buffers, preservatives, wetting agents, antioxidants, chelating agents, thickeners, gelling agents, pH adjusters and the like. Examples of the thickener include: HES, dextran, methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, and the like, but is not limited thereto. The concentration of the thickener varies with the thickener selected, and can be arbitrarily set within a range of concentrations that are safe and that achieve the desired viscosity when administered to a patient or subject.

The pharmaceutical composition of the present invention may further comprise 1 or more other drugs in addition to the adherent stem cells. Examples of the other drugs include: antibiotics, albumin preparations, vitamin preparations, anti-inflammatory agents, and the like, but are not limited thereto. As the above-mentioned anti-inflammatory agent, there may be mentioned: 5-aminosalicylic acid preparations, steroid preparations, immunosuppressants, biologics, etc., but are not limited thereto. Examples of the above 5-aminosalicylic acid preparation include: sulfasalazine, mesalamine, and the like, but are not limited thereto. Examples of the steroid preparation include: cortisone, prednisolone, methylprednisolone, and the like, but are not limited thereto. Examples of the immunosuppressive agent include: tacrolimus, cyclosporine, methotrexate, azathioprine, 6-mercaptopurine, and the like, but is not limited thereto. Examples of the biological agent include: infliximab (infliximab), adalimumab (adalimumab), uitlizumab (ustekinumab), secukinumab (secukinumab), epratuzumab (ixekizumab), brodalumab (brodalumab), tocilizumab (tocilizumab), vedolizumab (vedolizumab), felodib (filigonitinib), golimumab (golimumab), polyethylene glycol-conjugated certolizumab (certolizumab pegol), abasic (abatacept), etanercept (etanercept), and the like, but is not limited thereto. In addition, other drugs mentioned above may be other types of cells.

In addition, the other drugs mentioned above may be other cells that can be administered. Examples of other cells to be administered include: blood-derived cells (leukocytes, erythrocytes, monocytes, etc.), vascular endothelial cells, vascular endothelial progenitor cells, vascular wall cells, pericytes, fibroblasts, skeletal muscle myoblasts, epithelial cells, stromal cells, mature adipocytes, organ parenchymal cells (liver parenchymal cells, cardiac muscle cells, etc.), nerve cells, and the like, but are not particularly limited.

The pH of the pharmaceutical composition of the present invention may be a pH around neutral, for example, pH5.5 or more, pH6.0 or more, pH6.5 or more, or pH7.0 or more, or pH10.5 or less, pH9.5 or less, pH8.5 or less, or pH8.0 or less, but is not limited thereto.

The cell concentration of the pharmaceutical composition of the present invention is a cell concentration at which a therapeutic effect on a disease can be obtained when the pharmaceutical composition is administered to a patient or a subject, as compared with a patient or a subject to which the pharmaceutical composition is not administered, and the specific cell concentration can be appropriately determined depending on the administration method, the purpose of use, the age, weight, symptoms and the like of the patient or the subject, and the lower limit of the cell concentration of the pharmaceutical composition of the present invention is not particularly limited, and is, for example, 1.0 × 10⁵1.0 × 10 of more than one/mL⁶1.2 × 10 of more than one/mL⁶1.4 × 10 of more than one/mL⁶1.6 × 10 of more than one/mL⁶1.8 × 10 of more than one/mL⁶2.0 × 10 of more than one/mL⁶More than 3.0 × 10⁶More than one/mL, 4.0 × 10⁶5.0 × 10 of more than one/mL⁶More than one/mL, 6.0 × 10⁶More than one/mL, 7.0 × 10⁶More than one/mL, 8.0 × 10⁶More than 9.0 × 10⁶More than 9.5 × 10⁶More than one/mL, or 1.0 × 10⁷The upper limit of the cell concentration of the pharmaceutical composition of the present invention is not particularly limited, but is, for example, 1.0 × 10¹⁰Less than 1/mL and 1.0 × 10⁹Less than 8.0 × 10 pieces/mL⁸Less than 6.0 × 10 pieces/mL⁸Less than 4.0 × 10 pieces/mL⁸2.0 × 10 below one/mL⁸Less than or equal to 1.0 × 10⁸Less than one/mL.

The pharmaceutical composition of the present invention is preferably a liquid preparation, more preferably a liquid preparation for injection. As the liquid preparation for injection, for example, liquid preparations suitable for injection are known from international publication No. WO2011/043136, japanese patent application laid-open No. 2013-256510 and the like. The pharmaceutical composition of the present invention can be prepared into a liquid preparation for injection as described in the above-mentioned documents.

The liquid preparation may be a suspension of cells or a liquid preparation in which cells are dispersed in the liquid preparation. The form of the cells contained in the liquid preparation is not particularly limited, and may be, for example, single cells or cell aggregates.

In the case where the pharmaceutical composition of the present invention is a liquid preparation for injection, the lower limit of the cell concentration of the liquid preparation for injection is preferably 1.0 × 10 from the viewpoint of enhancing the therapeutic effect on diseases⁶1.2 × 10 of more than one/mL⁶1.4 × 10 of more than one/mL⁶1.6 × 10 of more than one/mL⁶1.8 × 10 of more than one/mL⁶2.0 × 10 of more than one/mL⁶More than 3.0 × 10⁶More than one/mL, 4.0 × 10⁶5.0 × 10 of more than one/mL⁶More than one/mL, 6.0 × 10⁶More than one/mL, 7.0 × 10⁶More than one/mL, 8.0 × 10⁶More than 9.0 × 10⁶More than 9.5 × 10⁶More than one/mL, or 1.0 × 10⁷One or more per mL, the upper limit of the cell concentration of the liquid preparation for injection is preferably 1.0 × 10 from the viewpoint of ease of preparation and administration of the liquid preparation for injection⁹Less than 8.0 × 10 pieces/mL⁸Less than 6.0 × 10 pieces/mL⁸Less than 4.0 × 10 pieces/mL⁸2.0 × 10 below one/mL⁸Less than or equal to 1.0 × 10⁸Less than one/mL.

In addition, according to one embodiment of the present invention, the pharmaceutical composition of the present invention may be a preparation for transplantation of a cell mass or a sheet-like structure. For example, as a preparation for transplantation having a cell mass structure, a preparation for transplantation containing a cell mass obtained by binding separated cells with a binder (e.g., fibrinogen) is known in international publication No. WO 2017/126549. Further, as a preparation for transplantation having a sheet-like structure, for example, a cell sheet obtained by culturing using a temperature-responsive culture dish (for example, UpCell (registered trademark) (manufactured by CellSeed corporation)), a laminate of a sheet-like cell culture and fibrin gel, a cell-coated sheet obtained by coating a cell suspension on a sheet-like base material, and the like are known in international publication No. WO2006/080434, japanese patent application laid-open No. 2016-52272. The pharmaceutical composition of the present invention can be prepared into preparations for transplantation of various cell aggregates or sheet-like structures by using, for example, the methods described in the above-mentioned documents.

In addition, according to one embodiment of the present invention, the pharmaceutical composition of the present invention may be a gel preparation in which cells are mixed with an arbitrary gel. As the gel preparation, for example, a cell therapeutic agent containing an adherent stem cell-hydrogel composition as an active ingredient is known in japanese patent publication No. 2017-529362. The pharmaceutical composition of the present invention can be prepared into a gel preparation as described in the above-mentioned documents.

The method of administering the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include: subcutaneous injection, intradermal injection, intramuscular injection, intralymphatic injection, intravenous injection, intraarterial injection, intraperitoneal injection, intrathoracic injection, direct local injection, direct sticking, or direct local transplantation, etc. According to one embodiment of the present invention, the liquid preparation for injection can be filled in a syringe and administered through an injection needle or a catheter into a vein, an artery, a myocardium, a joint cavity, a hepatic artery, a muscle, an epidural, a gum, a ventricle, a subcutaneous tissue, an intradermal tissue, an abdominal cavity, or a portal vein, but the present invention is not limited thereto. As for the method of administration of the pharmaceutical composition, for example, in Japanese patent laid-open No. 2015-61520, Onken JE, t al.American College of gastroenterology Conference 2006 Las Vegas, NV, Abstract 121, Garcia-Olmo D, et al.Dis Colon Rectum 2005; 48:1416-23, etc. intravenous injection, intravenous drip, direct local injection, direct local transplantation, etc. are known. The pharmaceutical composition of the present invention can be administered by various methods described in the above documents.

The dose of the pharmaceutical composition of the present invention is an amount of cells which can obtain a therapeutic effect on a disease in comparison with a patient or a subject to which the pharmaceutical composition is not administered when administered to the patient or the subject, and the specific dose can be appropriately determined depending on the administration form, the administration method, the purpose of use, the age, weight, symptoms and the like of the patient or the subject, and the dose of 1 dose of human adherent stem cells is not particularly limited, and is, for example, 1 × 10⁴1 × 10 above body weight/kg⁵5 × 10 above per kg body weight⁵1 × 10 above body weight/kg⁶2 × 10 of more than one/kg body weight⁶4 × 10 above body weight/kg⁶6 × 10/kg body weight of the human body⁶More than or equal to 8 × 10 per kg body weight⁶The dose per kg body weight of the human adherent stem cells is not particularly limited to 1 dose, and is, for example, 1 × 10¹²Less than 1/kg body weight, 1 × 10¹¹Less than 1/kg body weight, 1 × 10¹⁰Less than 1/kg body weight, 1 × 10⁹Less than 5/kg body weight, 5 × 10⁸Less than 1/kg body weight, 1 × 10⁸Less than 8/kg body weight, 8 × 10⁷Less than 6/kg body weight, 6 × 10⁷Less than 4 × 10 per kg body weight⁷Less than 2/kg body weight, or 2 × 10⁷Less than one/kg body weight.

In the case where the pharmaceutical composition of the present invention is a liquid preparation for injection, the 1 dose of the liquid preparation for injection to human adherent stem cells is preferably 1 × 10 from the viewpoint of enhancing the therapeutic effect on diseases⁵5 × 10 above per kg body weight⁵1 × 10 above body weight/kg⁶2 × 10 of more than one/kg body weight⁶4 × 10 above body weight/kg⁶6 × 10/kg body weight of the human body⁶More than or equal to 8 × 10 per kg body weight⁶More than one per kg body weight, and from the viewpoint of ease of preparation and administration of the liquid preparation for injection, 1 dose of the liquid preparation for injection to human adherent stem cells is preferably 1 × 10⁹Less than 5/kg body weight, 5 × 10⁸Less than 1/kg body weight, 1 × 10⁸Less than 8/kg body weight, 8 × 10⁷Less than 6/kg body weight, 6 × 10⁷Less than 4 × 10 per kg body weight⁷Less than 2/kg body weight, or 2 × 10⁷Less than one/kg body weight.

The frequency of administration of the pharmaceutical composition of the present invention is a frequency at which a therapeutic effect on a disease can be obtained when the pharmaceutical composition is administered to a patient or a subject. The specific administration frequency can be determined appropriately according to the administration form, administration method, purpose of use, and age, body weight, symptoms, and the like of the patient or subject, and is, for example, 1 time for 4 weeks, 1 time for 3 weeks, 1 time for 2 weeks, 1 time for 1 week, 2 times for 1 week, 3 times for 1 week, 4 times for 1 week, 5 times for 1 week, 6 times for 1 week, or 7 times for 1 week.

The period of administration of the pharmaceutical composition of the present invention is a period in which a therapeutic effect on a disease can be obtained when the pharmaceutical composition is administered to a patient or a subject. The specific administration period may be appropriately determined depending on the administration form, administration method, purpose of use, and age, body weight, symptoms, and the like of the patient or subject, and is, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.

The timing of administering the pharmaceutical composition of the present invention to a patient or a subject is not particularly limited, and examples thereof include: immediately after onset, within n days from onset (n represents an integer of 1 or more), immediately after diagnosis, within n days from diagnosis (n represents an integer of 1 or more), before remission, during remission, after remission, before relapse, during relapse, after relapse, and the like.

The pharmaceutical composition of the present invention may be stored in a frozen state until just before use. The temperature for cryopreservation is preferably-30 deg.C or lower, -40 deg.C or lower, -50 deg.C or lower, -80 deg.C or lower, -90 deg.C or lower, -100 deg.C or lower, -150 deg.C or lower, -180 deg.C or lower, or-196 deg.C or lower (liquid nitrogen temperature). When the pharmaceutical composition of the present invention is administered to a patient or a subject, it can be used by rapid thawing at 37 ℃.

The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples.

Examples

< comparative example 1: research on index >

In comparative example 1 and example 1 shown below, an index for obtaining a cell population including adherent stem cells exhibiting high immunosuppressive action and/or proliferation was examined.

(step 1: collection of amnion)

Fetal membranes and placenta were aseptically collected as fetal appendages from pregnant women (donor a) who had received informed consent for cases of elective caesarean delivery. The obtained fetal membranes and placenta were placed in a sterilized pot containing physiological saline, and the amnion was manually peeled off from the cut ends of the fetal membranes. The amniotic membrane was washed with Hank's balanced salt solution (Ca/Mg free) to remove adherent blood and blood clots.

(step 2: enzyme treatment of amnion and recovery of amnion MSC)

Amnion obtained from donor a was immersed in Hank's balanced salt solution (containing Ca/Mg) containing 240PU/mL collagenase and 200PU/mL Dispase I, and subjected to enzyme treatment by shaking at 37 ℃ for 90 minutes at 50 rpm. The enzyme-treated solution was filtered through a nylon mesh having a mesh size of 95 μm to remove undigested amnion, and a cell suspension containing amnion MSCs was recovered.

The obtained cell suspension was analyzed by flow cytometry for the ratio of cells positive for expression of CD90, which is one of surface antigens known as representative positive markers of MSCs, and it was confirmed that amniotic MSCs were isolated from amnion at high purity.

Surface antigen analysis was performed using a BD Accuri of Becton, Dickinson and Company (BD)^TMC6Flow Cytometer, after analyzing cell number: 10,000cells, flow rate settings: slow (14. mu.L/min). In this assay, FITC Mouse IgG1,. kappa.Isotype Control (manufactured by BD Co., Ltd.: 550616) was used as an antibody for Isotype Control, and FITC Mouse Anti-Human CD90 (manufactured by BD Co., Ltd.: 555595) was used as an antibody for CD90 antigen.

(step 3: culture of amniotic membrane MSC)

The above-mentioned "step 2: a part of the cell population containing amniotic membrane MSCs obtained in the step of subjecting amniotic membrane to enzyme treatment and recovering amniotic membrane MSCs was 6000 cells/cm²The density of (A) was inoculated into a culture vessel CellSTACK (registered trademark) (manufactured by Corning Co., Ltd.)) Adherent culture was performed to near confluence in α MEM (alpha modification of Minimum Essential Medium eagle) containing 10% bovine fetal serum (FBS) (inactivated) and 10ng/mL basic fibroblast growth factor (bFGF) at final concentration.

Cell clusters at passage 0 were detached using TrypLE Select (manufactured by Thermo Fisher Scientific), and cell clusters of 1/5 amount were inoculated to CellSTACK (registered trademark) for subculture. This subcultured cell population was referred to as a 1 st passage cell population.

At the time of reaching near confluence, the cell population of passage 1 was detached using TrypLE Select, diluted with medium, and recovered by centrifugation. The recovered cell population was suspended in RPMI 1640. To this, an equal amount of a CP-1 (registered trademark) solution (a mixture of CP-1 (registered trademark) and 25% human serum albumin in a ratio of 34: 16) (manufactured by Jidong pharmaceutical industries, Ltd.) was added, and the mixture was slowly frozen to-80 ℃ and then frozen in liquid nitrogen. Then, unfreezing is carried out at the speed of 15000-18000 pieces/cm²Was inoculated into CellSTACK (registered trademark), and subcultured to near confluence. This subcultured cell population was referred to as a 2 nd passage cell population.

The cell population at passage 2 was detached using TrypLE Select, and 1/5 cells were inoculated into CellSTACK (registered trademark) for subculture. This subcultured cell population was referred to as a 3 rd passage cell population.

At the time of reaching near confluence, the cell population of passage 3 was detached using TrypLE Select, diluted with medium, and recovered by centrifugation. The recovered cell population was suspended in RPMI 1640. An equal amount of CP-1 (registered trademark) solution was added thereto, slowly frozen to-80 ℃ and then cryopreserved in liquid nitrogen. Then, thawing was carried out at 6000 cells/cm²Was inoculated into CellSTACK (registered trademark), and subcultured to near confluence. This subcultured cell population was referred to as a 4 th passage cell population.

The cell population at passage 4 was detached using TrypLE Select, and 1/5 cells were inoculated into CellSTACK (registered trademark) for subculture. This subcultured cell population was referred to as a 5 th passage cell population.

At the time of reaching near confluence, the cell population of the 5 th passage was detached using TrypLE Select, diluted with a medium, and recovered by centrifugation. The recovered cell population was suspended in RPMI 1640. An equal amount of CP-1 (registered trademark) solution was added thereto, slowly frozen to-80 ℃ and then cryopreserved in liquid nitrogen.

(step 4: surface antigen analysis)

The cell population at passage 5 of comparative example 1 was measured for each surface antigen (positive rate of CD73, positive rate of CD90, positive rate of CD105, positive rate and negative rate of CD 45) using a flow cytometer.

Surface antigen analysis was performed using a BD Accuri of Becton, Dickinson and Company (BD)^TMC6Flow Cytometer, after analyzing cell number: 10,000cells, flow rate settings: slow (14. mu.L/min). In this measurement, FITC Mouse IgG1,. kappa.Isotype Control (BD Co./model: 550616) was used as an antibody against the CD73 antigen, FITC Mouse Anti-Human CD73(BD Co./model: 561254) was used as an antibody against the CD90 antigen, FITC Mouse Anti-Human CD90(BD Co./model: 555595) was used as an antibody against the CD105 antigen, Anti-Human Antibodies FITC Conjugate (BioLegend Co./model: 323203) was used as an antibody against the CD45 antigen, and FITC Mouse Anti-Human CD45(BD Co./model: 555482) was used as an antibody against the CD45 antigen.

As a result of the surface antigen analysis, in the cell population at passage 5 of comparative example 1, the positive rates of CD73, CD90 and CD105 were all 90% or more (specifically, CD 73: 100%, CD 90: 100%, CD 105: 98%), and the positive rate of CD45 was less than 5% (negative rate: 95% or more) (specifically, positive rate of CD 45: 0% (negative rate: 100%). From the above results, it was confirmed that the cell population at passage 5 of comparative example 1 was a cell population including adherent stem cells.

(step 5: analysis of Gene expression)

The expression of PLIN2 gene, NEFM gene, and SDHA gene was analyzed by quantitative RT-PCR on the cell population at passage 5 of comparative example 1. The quantitative RT-PCR was specifically carried out in accordance with the following procedure A (comparative Ct method).

(step A: comparative Ct method)

(4) The PCR reaction was carried out using KOD SYBR qPCR Mix (manufactured by Toyo Boseki Co., Ltd.) with the synthesized cDNA as a template. The liquid volume of the reaction solution was 20. mu.L. The PCR conditions were 40 cycles (98 ℃ for 10 seconds, 60 ℃ for 10 seconds, and 68 ℃ for 30 seconds) using a StepOneNusReal-Time PCR System (Thermo Fisher Scientific Co., Ltd.) maintained at 98 ℃ for 2 minutes.

(5) The relative expression amount (X) of the gene to be measured with respect to the gene to be compared is represented by X ═ 2^{-(Y-Z)}And (4) calculating. Here, Y is a Ct value of a gene to be measured, and Z is a Ct value of a gene to be compared.

As a result, the relative expression level of PLIN2 gene with respect to the expression level of SDHA gene was 1.11 and the relative expression level of NEFM gene with respect to the expression level of SDHA gene was 0.04 for the cell population at passage 5 of comparative example 1. From the above, it was found that the cell population at the 5 th passage of comparative example 1 did not satisfy the condition (b) shown below.

(b) The relative expression level of the PLIN2 gene to the expression level of the SDHA gene was 1.50 or more.

In the case of quantitative RT-PCR according to the following procedure B (calibration curve method) on the cell population at passage 5 of comparative example 1, the relative expression level of PLIN2 gene to SDHA gene was 0.27, and the relative expression level of NEFM gene to SDHA gene was 1.51.

(step B: calibration Curve method)

(3) cDNA was synthesized by reverse transcription reaction using the purified total RNA as a template and ReverTra Ace qPCR RT Master Mix with DNA Remover (Toyo chemical Co., Ltd.).

(4) PCR was carried out using the synthesized cDNA as a template and Power SYBR Green PCR Master Mix (manufactured by ThermoFisher Scientific Co., Ltd.). The liquid volume of the reaction solution was 20. mu.L. The PCR conditions were 60 cycles (95 ℃ for 3 seconds and 60 ℃ for 30 seconds) using a StepOneplus Real-Time PCR System (Thermo Fisher Scientific Co., Ltd.) maintained at 50 ℃ for 2 minutes and 95 ℃ for 2 minutes. As a sample for drawing a calibration curve, a sample obtained by diluting the synthesized cDNA at a level of 1 to 5 times (double-fold) was used.

(6) The relative expression level (W) of the target gene to be compared was calculated from W ═ α/β. Here, α is the expression level of the gene to be measured, and β is the expression level of the gene to be compared.

(step 6: evaluation of expression level of TNFAIP6 Gene)

The expression level of TNFAIP6 gene in the cell population of the 5 th passage of comparative example 1 was evaluated by using a microarray. As a result, the relative expression amount of TNFAIP6 gene with respect to the expression amount of SDHA gene in the cell population at passage 5 of comparative example 1 was 0.41.

(step 7: evaluation of growth inhibition Rate of hPPBMC)

The immunosuppressive effects of the 5 th passage cell population of comparative example 1 were evaluated by a mixed lymphocyte reaction test (MLR test). As a result, the proliferation inhibition ratio of hBMC in the cell population of the 5 th passage of comparative example 1 was 42.0%.

(step 8: evaluation of specific growth Rate)

The specific growth rate of the cell population at the 5 th passage in comparative example 1 and example 1 was evaluated. As a result, the specific growth rate of the cell population of comparative example 1 was 0.24 (1/day).

< example 1: research on index >

(step 1: collection of amnion)

An amnion was obtained in the same manner as in step 1 of comparative example 1, except that a fetal membrane and a placenta as fetal appendages were aseptically collected from the same donor (donor a) as in comparative example 1 and a donor (donor B) different from the donor of comparative example 1.

(step 2: enzyme treatment of amnion and recovery of amnion MSC)

A cell suspension containing amniotic MSCs was recovered by the same method as in step 2 of comparative example 1. The percentage of cells expressing CD90 in the obtained cell suspension was analyzed as in comparative example 1, and it was confirmed that amniotic MSCs could be isolated from amniotic membrane at high purity.

(step 3: culture of amniotic membrane MSC)

A cell population of passage 5 was obtained in the same manner as in step 3 of comparative example 1, except that α MEM containing 5% human platelet lysate (hPL) was used instead of α MEM containing 10% FBS and 10ng/mL bFGF.

(step 4: surface antigen analysis)

Surface antigen analysis of the cell population at passage 5 of example 1 was performed by the same method as in step 4 of comparative example 1. As a result, in the cell population at passage 5 from donor A in example 1, the positive rates of CD73, 90 and 105 were all 90% or more (specifically, CD 73: 98%, CD 90: 100% and CD 105: 100%), and the positive rate of CD45 was less than 5% (negative rate: 95% or more) (specifically, positive rate of CD 45: 0% (negative rate: 100%). In addition, in the cell population at passage 5 from donor B in example 1, the positive rates of CD73, 90, and 105 were all 90% or more (specifically, CD 73: 100%, CD 90: 99%, CD 105: 98%), and the positive rate of CD45 was less than 5% (negative rate: 95% or more) (specifically, positive rate of CD 45: 0% (negative rate: 100%). From the above results, it was confirmed that the cell population at passage 5 of example 1 was a cell population containing adherent stem cells.

(step 5: analysis of Gene expression)

The gene expression analysis of the cell population at passage 5 of example 1 was performed according to step a (comparative Ct method) described in step 5 of comparative example 1. The relative expression levels of the respective genes are shown in table 1.

TABLE 1 relative expression amounts of the respective genes

As shown in Table 1, in the cell population at passage 5 from donor A of example 1, the relative expression amount of PLIN2 gene with respect to the expression amount of SDHA gene was 1.95, the relative expression amount of NEFM gene with respect to the expression amount of SDHA gene was 0.0006, and the relative expression amount of PLIN2 gene with respect to the expression amount of NEFM gene was 3051. In addition, in the cell population at passage 5 from donor B of example 1, the relative expression level of the PLIN2 gene with respect to the expression level of the SDHA gene was 5.32, the relative expression level of the NEFM gene with respect to the expression level of the SDHA gene was 0.017, and the relative expression level of the PLIN2 gene with respect to the expression level of the NEFM gene was 315.

From this, it was found that the cell population at passage 5 of example 1 satisfied the conditions (a) and (b) shown below.

When gene expression analysis was performed according to step B (calibration curve method) described in step 5 of comparative example 1, the relative expression amounts of the respective genes in the cell population at passage 5 of example 1 are shown in table 2.

TABLE 2 relative expression amounts of the respective genes with respect to the expression amount of SDHA Gene

(step 6: evaluation of expression level of TNFAIP6 Gene)

The expression level of TNFAIP6 gene in the 5 th passage cell population derived from donor A of example 1 was evaluated in the same manner as in step 6 of comparative example 1. As a result, the relative expression amount of TNFAIP6 gene with respect to the expression amount of SDHA gene in the cell population of 5 th passage derived from donor a in example 1 was 1.96. As shown above, the cell population at passage 5 from donor a of example 1 highly expressed TNFAIP6 gene, which is a gene encoding an immunosuppressive-associated cytokine, and showed high immunosuppressive effect.

(step 7: evaluation of growth inhibition Rate of hPPBMC)

The immunosuppressive effect of the 5 th passage cell population derived from donor a in example 1 was evaluated by the same method as in step 7 of comparative example 1. As a result, the hBMC proliferation inhibition rate of the cell population at passage 5 from donor A in example 1 was 54.4%. From the above, it was found that the cell population of the 5 th passage derived from donor A in example 1 significantly inhibited the proliferation of hBMC activated by PHA stimulation, and showed high immunosuppressive activity.

From these results, it was found that the cell population having the cell characteristics of (a) and (b) showed a high immunosuppressive activity. Furthermore, it was shown that the conditions (a) and (b) described above are effective as an index for obtaining a cell population containing adherent stem cells exhibiting a high immunosuppressive effect.

(step 8: evaluation of specific growth Rate)

The specific growth rate of the cell population at the 5 th passage of example 1 was evaluated in the same manner as in step 8 of comparative example 1. The results of measuring the specific growth rate are shown in Table 3.

TABLE 3 specific growth rates of the respective cell populations of comparative example 1 and example 1

As shown in Table 3, the specific growth rate of the cell population derived from donor A in example 1 was 0.55(1/day), and the specific growth rate of the cell population derived from donor B in example 1 was 0.87 (1/day). From the above, it was found that the cell population at passage 5 of example 1 showed high proliferation properties.

From these results, it was found that the cell population having the cell characteristics of (a) and (b) showed high proliferation properties. Furthermore, it was shown that the conditions (a) and (b) described above are effective as an index for obtaining a cell population containing adherent stem cells exhibiting high proliferation.

From the above results, it was found that the cell population having the cell characteristics of (a) and (b) showed not only a high immunosuppressive action but also a high proliferative property. In addition, the conditions (a) and (b) are effective as an index for obtaining a cell population containing adherent stem cells exhibiting high immunosuppressive action and/or proliferation. That is, according to the present invention, by using the conditions of (a) and (b) as an index, a cell population including adherent stem cells exhibiting high immunosuppressive action and/or proliferation properties can be obtained, whereby the cell culture period can be shortened and the production cost can be reduced.

< example 2: study on hPPBMC-induced immunosuppression in GVHD mice

In example 2 shown below, it was examined whether or not a cell population having the cell characteristics of the following (a) and (b) shows an immunosuppressive effect in an animal experiment.

(step 1: collection of amnion)

An amnion was obtained in the same manner as in step 1 of comparative example 1, except that a fetal membrane and a placenta as fetal appendages were aseptically collected from a donor (donor E) different from the donor of comparative example 1 and example 1.

(step 2: enzyme treatment of amnion and recovery of amnion MSC)

A cell suspension containing amniotic MSCs was recovered by the same method as in step 2 of comparative example 1.

(step 3: culture of amniotic membrane MSC)

A cell population was obtained by the same method as in step 3 of example 1.

(step 4: analysis of Gene expression)

Gene expression analysis of the cell population at passage 5 from the donor E was performed according to the same procedure a (comparative Ct method) as in step 5 of comparative example 1. The relative expression levels of the respective genes are shown in Table 4.

TABLE 4 relative expression amounts of the respective genes

As shown in Table 4, the relative expression level of PLIN2 gene with respect to the expression level of SDHA gene, the relative expression level of NEFM gene with respect to the expression level of SDHA gene was 7.94, and the relative expression level of PLIN2 gene with respect to the expression level of NEFM gene was 20343 for the cell population at passage 5 from donor E.

From this, it was found that the cell population at the 5 th passage derived from the donor E satisfied the conditions (a) and (b) shown below.

As shown in table 4, the relative expression amount of the NEFM gene with respect to the SDHA gene in the cell population at passage 5 from the donor E was less than 0.02.

As shown in Table 4, the relative expression level of PLIN2 gene with respect to the expression level of NEFM gene in the cell population at passage 5 derived from donor E was 250 or more.

(step 5: evaluation of growth inhibition Rate of hPPBMC)

The immunosuppressive effect of the 5 th passage cell population from donor E was evaluated by mixed lymphocyte reaction assay (MLR assay). As a result, the growth inhibition ratio of hBMC in the above cell population was 53.2%. From the above, it was found that the cell population at passage 5 derived from donor a in example 1 inhibited the proliferation of hPBMC activated by PHA stimulation, and showed immunosuppressive action.

(step 6: evaluation of the immunosuppressive Effect of GVHD mice induced by Using hPPMC)

In mice induced with Graft Versus Host Disease (GVHD) using human peripheral blood mononuclear cells (hPBMC) (hPBMC-induced GVHD mice), the immunosuppressive effect of the 5 th passage cell population derived from donor E was evaluated.

Specifically, 3.0 × 10 10.8 mL/min caudal vein administration was performed on 7-8-week-old male mice of NOD/Shi-scid, IL-2 Ry KO (NOG) line, in particular, 3.0 × 10.2 mL/min⁶0.2mL/Head of hPPMC. The date of administration of hBMC was taken as day 0.

The cryopreserved cell population from the 5 th passage of donor E was thawed, diluted with physiological saline, and centrifuged to remove a portion of the supernatant to prepare 1.6 × 10⁶The cell population was resuspended at one/mL, and the hPGMC-induced GVHD mice prepared using the above hPGMC were administered to the above cell population via the caudal vein at a rate of 1.2 mL/min, at an amount of 8.0 × 10⁶One/5 mL/kg body weight. The administration frequency was 3 times, the administration on days 7, 14 and 21 in the early administration group, and the administration on days 21, 28 and 35 in the late administration group. The control group was hPBMC-induced GVHD mice not administered with the above cell population.

For each of the above groups, survival rate was evaluated. For the evaluation of the survival rate, a survival time analysis (Log-RANK test) was performed. In addition, for the evaluation of survival rate, the case where the significance level (risk factor, risk rate) was less than 5% (p < 0.05) was judged as significant difference.

The results of survival rate are shown in fig. 1.

As a result, the survival rate of hPBMC-induced GVHD mice in the former administration group was significantly improved compared to the control group. In the post-administration group, the survival rate of hPBMC-induced GVHD mice showed an improved tendency relative to the control group. In the former group, hPBMC induced weight loss in GVHD mice tended to be suppressed compared to the control group. This indicates that the 5 th passage cell population from donor E improved hPPBMC-induced GVHD in GVHD mice.

From these results, it was found that the cell population having the cellular characteristics of (a) and (b) showed immunosuppressive effects not only in the mixed lymphocyte reaction test (MLR test), but also in the hBMC-induced GVHD mice. This indicates that the conditions (a) and (b) described above are effective as an index for obtaining a cell population containing adherent stem cells exhibiting a high immunosuppressive effect.

(step 7: evaluation of specific growth Rate)

The specific proliferation rate of the cell population from the 5 th passage of donor E was evaluated. As a result, the specific growth rate of the cell population at the 5 th passage from the donor E was 0.44 (1/day).

Therefore, it was found that the cell population having the cell characteristics of (a) and (b) showed high proliferation properties. Furthermore, it was shown that the conditions (a) and (b) described above are effective as an index for obtaining a cell population containing adherent stem cells exhibiting high proliferation.

The results of example 2 above are summarized in table 5.

TABLE 5 summary of example 2

As is clear from Table 5, the cell population having the cellular characteristics of (a) and (b) showed high immunosuppressive activity and high proliferation activity in the mixed lymphocyte reaction test (MLR test) and the hBMC-induced GVHD mice. Furthermore, it was shown that the conditions (a) and (b) described above are effective as an index for obtaining a cell population containing adherent stem cells exhibiting high immunosuppressive activity and/or proliferation. That is, according to the present invention, by using the conditions of (a) and (b) as an index, a cell population including adherent stem cells exhibiting high immunosuppressive action and/or proliferation properties can be obtained, whereby the cell culture period can be shortened and the production cost can be reduced.

< example 3: preparation of pharmaceutical composition

A portion of the 5 th passage cell population of example 1 was used for the preparation of a pharmaceutical composition, preparation contained 2.0 × 10⁸Adherent stem cells, 6.8mL of CP-1 (registered trademark) solution, 3.2mL of 25% human serum albumin solution, and 10mL of RPMI1640 medium. The pharmaceutical composition is sealed in a freezing bag and stored in a frozen state. In addition, the pharmaceutical composition may be thawed at the time of use and administered to a patient or subject.

< example 4: preparation of amnion-derived adherent Stem cells

Cell populations of passage 2 were obtained in the same manner as in steps 1 to 3 of example 1, except that fetal membranes were collected from 2 donors (donor F, G) different from those of comparative example 1, example 1 and example 2.

Comparative example 2: preparation of bone marrow-derived adherent Stem cells

The whole amount of commercially available bone marrow fluid (AllCells Corp./model: ABM003) was inoculated, and primary culture was carried out in α MEM (alpha Modification of Minimum essential Medium eagle) containing 10% bovine fetal serum (FBS) (inactivated) in final concentration.A part of the 0 th passage cell population containing the obtained bone marrow-derived adherent stem cells was cultured at 6000 cells/cm²The density of (A) was inoculated into CellSTACK (registered trademark) (manufactured by Corning Co., Ltd.), and the mixture was added to α MEM containing 10% FBS (inactivated) in terms of final concentrationSubculture was performed. The cell population containing the obtained bone marrow-derived adherent stem cells was subjected to cryopreservation in a cell preservation solution containing a CP-1 (registered trademark) solution.

< example 5: surface antigen analysis

(surface antigen analysis)

The cell populations obtained in example 4, comparative example 1, and comparative example 2 were analyzed for each surface antigen (CD73, CD90, CD105, CD45, and CD326) using flow cytometry dot plot analysis. The flow cytometry dot chart analysis method is as described in the above steps (1) to (8).

The ratio of cells positive for each surface antigen is shown in table 6.

TABLE 6 ratio of cells positive for each surface antigen

As shown in table 6, the positive rates of CD73, CD90, and CD105 were all 90% or more, and the positive rates of CD45 and CD326 were less than 5% (negative rate was 95% or more) in each cell population.

< example 6: evaluation of Gene expression level

The gene expression analysis of the cell population obtained in example 4, comparative example 1 and comparative example 2 was performed according to step a (comparative Ct method) described in step 5 of comparative example 1. The relative expression levels of the respective genes are shown in Table 7.

TABLE 7 relative expression amounts of the respective genes

As shown in Table 7, the expression of the PLIN2 gene and NEFM gene was positive in the amnion cell population-F, G, A and bone marrow cell population. The relative expression levels of PLIN2 gene in each of the amniotic membrane cell populations-F and G with respect to SDHA gene were 1.50 or more in both of the cell populations (amniotic membrane cell population-F: 2.02 and amniotic membrane cell population-G: 2.12). On the other hand, the relative expression level of PLIN2 gene in the amniotic membrane cell population-A and the bone marrow cell population relative to the SDHA gene was less than 1.50 (amniotic membrane cell population-A: 1.11, bone marrow cell population: 1.31). From the above, it was found that the amniotic membrane cell population-F, G satisfies the following conditions (a) and (b). On the other hand, it was found that the amnion cell population-A and the bone marrow cell population do not satisfy the condition (b) above.

As shown in table 7, the relative expression amount of NEFM gene in the amniotic membrane cell population-F, G and the bone marrow cell population was less than 0.02 relative to the SDHA gene. The relative expression amount of the NEFM gene of the amniotic membrane cell population-A relative to the SDHA gene is 0.02 or more.

As shown in Table 7, the relative expression level of the PLIN2 gene of the amniotic membrane cell population-F, G to the expression level of the NEFM gene was 250 or more. The relative expression level of PLIN2 gene relative to the expression level of NEFM gene in the amnion cell population-A and bone marrow cell population was less than 250.

< example 7: evaluation of hPPMC-induced immunosuppression in GVHD mice

In mice induced with Graft Versus Host Disease (GVHD) by human peripheral blood mononuclear cells (hPBMC) (hPBMC-induced GVHD mice), the immunosuppressive effects of the cell populations (amnion cell population-F, A and bone marrow cell population) obtained in example 4, comparative example 1 and comparative example 2 were evaluated.

Animal experiments were performed in the same manner as in step 6 of example 2, except that the number of administrations was changed from 3 to 4 (administration on days 7, 14, 21, and 28). The control group was hPBMC-induced GVHD mice not administered with the above cell population.

For each of the above groups, the change in survival rate was evaluated. For the evaluation of the survival rate, a survival time analysis (Log-RANK test) was performed. In addition, in the transition of survival rate, the case where the significance level (risk factor, risk rate) is less than 5% (p < 0.05) was judged as significant difference.

The results of survival rate are shown in fig. 2.

As shown in fig. 2, the survival rate of hPBMC-induced GVHD mice was significantly improved in the amnion cell population-F-administered group compared to the control group. In the amnion cell population-a administration group and the bone marrow cell population administration group, the survival rate of hPBMC-induced GVHD mice tended to be improved compared to the control group. In addition, in the amnion cell population-F-administered group, the hPBMC-induced weight loss in GVHD mice tended to be suppressed compared to the control group. As described above, the amniotic membrane cell population-F was found to have a higher effect of improving the GVHD induced by hPPMC than other cell populations (amniotic membrane cell population-A and bone marrow cell population). That is, the amniotic membrane cell population-F shows a high immune effect as compared with other cell populations. Thus, it was revealed that the cell population satisfying the conditions (a) and (b) showed a high immunosuppressive action. From this, it is considered that the conditions (a) and (b) are effective as an index for obtaining a cell population containing adherent stem cells exhibiting a high immunosuppressive action.

< example 8: quality evaluation of biological samples collected from different donors

In example 8 shown below, it was examined whether or not the quality of the donor itself and the biological sample collected from the donor can be evaluated by using the conditions (a) and (b) described below as indicators.

(step 1: collection of amnion)

An amnion was obtained in the same manner as in step 1 of comparative example 1, except that the fetal membrane and placenta as fetal appendages were aseptically collected from 2 donors (donor C, D) different from those of comparative example 1, example 2, and example 4.

(step 2: enzyme treatment of amnion and recovery of amnion MSC)

A cell suspension containing amniotic MSCs was recovered by the same method as in step 2 of example 1.

(step 3: culture of amniotic membrane MSC)

The cell population at passage 0 was obtained in the same manner as in step 3 of example 1.

(step 4: analysis of Gene expression)

Gene expression analysis of the 0 th passage cell population from each donor (donor C, D) was performed according to step a (comparative Ct method) described in step 5 of comparative example 1. As a result, the cell population at passage 0 derived from the donor C did not satisfy the conditions (a) and (b) described above. On the other hand, the cell population at passage 0 from the donor D satisfied the conditions (a) and (b) described above.

(step 5: evaluation of specific growth Rate)

The specific proliferation rate of the cell population from passage 0 of each donor (donor C, D) was evaluated. The results of measuring the specific growth rate are shown in Table 8.

TABLE 8 specific proliferation Rate of cell populations from passage 0 of each donor

As shown in Table 8, the specific growth rate of the cell population at the 0 th passage from the donor C was 0.18(1/day), and the specific growth rate of the cell population at the 0 th passage from the donor D was 0.46 (1/day). From the above, it was found that the cell population at passage 0 derived from the donor D showed high proliferation.

The above results show that even when collecting and culturing amnion MSCs from amnion by the same method, there is a great difference in the proliferation of the obtained cell population depending on the donor, and it is possible to evaluate the quality of the donor itself and the biological sample collected from the donor by examining the conditions of (a) and (b) above. That is, according to the present invention, by using the conditions (a) and (b) as indicators, the quality of the donor itself and the biological sample collected from the donor can be evaluated, and a biological sample having a high content of adherent stem cells having high proliferation can be selected (donor screening) in advance. This makes it possible to omit unnecessary cell culture or shorten the cell culture period, thereby reducing the production cost.

< example 9: monitoring during cultivation Process

In example 9 shown below, it was examined whether or not the immunosuppressive action and/or the proliferation property of adherent stem cells can be monitored by using the conditions (a) and (b) described below as an index in the culture step.

(step 3: culture of amniotic membrane MSC)

Since the 0 th passage cell population derived from the donor D obtained in step 3 of example 8 satisfies the conditions (a) and (b), the subsequent subculture is performed. The cell population was subcultured in the same manner as in step 3 of example 1 to obtain cell populations of 1 st to 5 th passages. Each subculture was performed to confirm whether or not the conditions (a) and (b) were satisfied. The 0 th passage of the cell population derived from the donor C obtained in step 3 of example 8 did not satisfy the conditions (a) and (b) described above, and therefore, the subsequent subculture was not performed.

(step 4: analysis of Gene expression)

Gene expression analysis of the cell population from the 1 st, 2 nd, 3 th, 4 th and 5 th passages of donor D was performed according to the procedure a (comparative Ct method) described in step 5 of comparative example 1. As a result, each cell population satisfies the conditions (a) and (b) described above.

(step 5: evaluation of specific growth Rate)

The specific proliferation rates of cell populations from 1 st, 2 nd, 3 th, 4 th and 5 th passages of donor D were evaluated. The results of measuring the specific growth rate of each cell population are shown in table 9.

TABLE 9 specific proliferation Rate of the respective cell populations from Donor D

As shown in Table 9, the specific growth rates of the cell populations at passages 1, 2, 3, 4 and 5 from the donor D were 0.64(1/day), 0.68(1/day), 0.41(1/day), 0.56(1/day) and 0.40(1/day), respectively. From the above, it was found that each cell population also showed high proliferation properties after subculture.

(step 6: evaluation of expression level of TNFAIP6 Gene)

The expression level of TNFAIP6 gene in the cell population from the 1 st, 2 nd, 3 th, 4 th and 5 th passages of donor D was evaluated. Each cell population expresses TNFAIP6 gene and has immunosuppressive effect.

As described above, it is possible to monitor the immunosuppressive action and/or proliferation of adherent stem cells by using the conditions (a) and (b) as indicators in the culture step. That is, according to the present invention, by measuring the above-mentioned index (preferably, by measurement over time), it is possible to quickly grasp and predict the change in the immunosuppressive action and/or the proliferative property of the adherent stem cells. This can shorten the cell culture period and reduce the production cost.

Sequence listing

<110> Kaneka CORPORATION clock (KANEKA CORPORATION)

<120> cell population comprising adherent stem cells, method for producing same, and pharmaceutical composition

<130>B17809WO01

<150>JP 2017-253878

<151>2017-12-28

<160>14

<170>PatentIn version 3.5

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Ile Thr Lys Ser Val Thr Val Thr Gln Lys Val Glu Glu His Glu Glu

500 505 510

acc ttt gag gag aaa cta gtg tct act aaa aag gta gaa aaa gtc act 1584

Thr Phe Glu Glu Lys Leu Val Ser Thr Lys Lys Val Glu Lys Val Thr

515 520 525

tca cac gcc ata gta aag gaa gtc acc cag agt gac taa 1623

Ser His Ala Ile Val Lys Glu Val Thr Gln Ser Asp

530 535 540

<210>4

<211>540

<212>PRT

<213> Intelligent people

<400>4

Met Ala Arg His Leu Arg Glu Tyr Gln Asp Leu Leu Asn Val Lys Met

1 5 10 15

Ala Leu Asp Ile Glu Ile Ala Ala Tyr Arg Lys Leu Leu Glu Gly Glu

20 25 30

Glu Thr Arg Phe Ser Thr Phe Ala Gly Ser Ile Thr Gly Pro Leu Tyr

35 40 45

Thr His Arg Pro Pro Ile Thr Ile Ser Ser Lys Ile Gln Lys Pro Lys

50 55 60

Val Glu Ala Pro Lys Leu Lys Val Gln His Lys Phe Val Glu Glu Ile

65 70 75 80

Ile Glu Glu Thr Lys Val Glu Asp Glu Lys Ser Glu Met Glu Glu Ala

85 90 95

Leu Thr Ala Ile Thr Glu Glu Leu Ala Val Ser Met Lys Glu Glu Lys

100 105 110

Lys Glu Ala Ala Glu Glu Lys Glu Glu Glu Pro Glu Ala Glu Glu Glu

115 120 125

Glu Val Ala Ala Lys Lys Ser Pro Val Lys Ala Thr Ala Pro Glu Val

130 135 140

Lys Glu Glu Glu Gly Glu Lys Glu Glu Glu Glu Gly Gln Glu Glu Glu

145 150 155 160

Glu Glu Glu Asp Glu Gly Ala Lys Ser Asp Gln Ala Glu Glu Gly Gly

165 170 175

Ser Glu Lys Glu Gly Ser Ser Glu Lys Glu Glu Gly Glu Gln Glu Glu

180 185 190

Gly Glu Thr Glu Ala Glu Ala Glu Gly Glu Glu Ala Glu Ala Lys Glu

195 200 205

Glu Lys Lys Val Glu Glu Lys Ser Glu Glu Val Ala Thr Lys Glu Glu

210 215 220

Leu Val Ala Asp Ala Lys Val Glu Lys Pro Glu Lys Ala Lys Ser Pro

225 230 235 240

Val Pro Lys Ser Pro Val Glu Glu Lys Gly Lys Ser Pro Val Pro Lys

245 250 255

Ser Pro Val Glu Glu Lys Gly Lys Ser Pro Val Pro Lys Ser Pro Val

260 265 270

Glu Glu Lys Gly Lys Ser Pro Val Pro Lys Ser Pro Val Glu Glu Lys

275 280 285

Gly Lys Ser Pro Val Ser Lys Ser Pro Val Glu Glu Lys Ala Lys Ser

290 295 300

Pro Val Pro Lys Ser Pro Val Glu Glu Ala Lys Ser Lys Ala Glu Val

305 310 315 320

Gly Lys Gly Glu Gln Lys Glu Glu Glu Glu Lys Glu Val Lys Glu Ala

325 330 335

Pro Lys Glu Glu Lys Val Glu Lys Lys Glu Glu Lys Pro Lys Asp Val

340 345 350

Pro Glu Lys Lys Lys Ala Glu Ser Pro Val Lys Glu Glu Ala Val Ala

355 360 365

Glu Val Val Thr Ile Thr Lys Ser Val Lys Val His Leu Glu Lys Glu

370 375 380

Thr Lys Glu Glu Gly Lys Pro Leu Gln Gln Glu Lys Glu Lys Glu Lys

385 390 395 400

Ala Gly Gly Glu Gly Gly Ser Glu Glu Glu Gly Ser Asp Lys Gly Ala

405 410 415

Lys Gly Ser Arg Lys Glu Asp Ile Ala Val Asn Gly Glu Val Glu Gly

420 425 430

Lys Glu Glu Val Glu Gln Glu Thr Lys Glu Lys Gly Ser Gly Arg Glu

435 440 445

Glu Glu Lys Gly Val Val Thr Asn Gly Leu Asp Leu Ser Pro Ala Asp

450 455 460

Glu Lys Lys Gly Gly Asp Lys Ser Glu Glu Lys Val Val Val Thr Lys

465 470 475 480

Thr Val Glu Lys Ile Thr Ser Glu Gly Gly Asp Gly Ala Thr Lys Tyr

485 490 495

Ile Thr Lys Ser Val Thr Val Thr Gln Lys Val Glu Glu His Glu Glu

500 505 510

Thr Phe Glu Glu Lys Leu Val Ser Thr Lys Lys Val Glu Lys Val Thr

515 520 525

Ser His Ala Ile Val Lys Glu Val Thr Gln Ser Asp

530 535 540

<210>5

<211>1851

<212>DNA

<213> Intelligent people

<220>

<221>CDS

<222>(1)..(1851)

<400>5

atg tcg ggg gtc cgg ggc ctg tcg cgg ctg ctg agc gct cgg cgc ctg 48

Met Ser Gly Val Arg Gly Leu Ser Arg Leu Leu Ser Ala Arg Arg Leu

1 5 10 15

gcg ctg gcc aag gcg tgg cca aca gtg ttg caa aca gga acc cga ggt 96

Ala Leu Ala Lys Ala Trp Pro Thr Val Leu Gln Thr Gly Thr Arg Gly

2025 30

ttt cac ttc act gtt gat ggg aac aag agg gca tct gct aaa gtt tca 144

Phe His Phe Thr Val Asp Gly Asn Lys Arg Ala Ser Ala Lys Val Ser

35 40 45

gat tcc att tct gct cag tat cca gta gtg gat cat gaa ttt gat gca 192

Asp Ser Ile Ser Ala Gln Tyr Pro Val Val Asp His Glu Phe Asp Ala

50 55 60

gtg gtg gta ggc gct gga ggg gca ggc ttg cga gct gca ttt ggc ctt 240

Val Val Val Gly Ala Gly Gly Ala Gly Leu Arg Ala Ala Phe Gly Leu

65 70 75 80

tct gag gca ggg ttt aat aca gca tgt gtt acc aag ctg ttt cct acc 288

Ser Glu Ala Gly Phe Asn Thr Ala Cys Val Thr Lys Leu Phe Pro Thr

85 90 95

agg tca cac act gtt gca gca cag cta gaa aat tat ggc atg ccg ttt 336

Arg Ser His Thr Val Ala Ala Gln Leu Glu Asn Tyr Gly Met Pro Phe

100 105 110

agc aga act gaa gat ggg aag att tat cag cgt gca ttt ggt gga cag 384

Ser Arg Thr Glu Asp Gly Lys Ile Tyr Gln Arg Ala Phe Gly Gly Gln

115 120 125

agc ctc aag ttt gga aag ggc ggg cag gcc cat cgg tgc tgc tgt gtg 432

Ser Leu Lys Phe Gly Lys Gly Gly Gln Ala His Arg Cys Cys Cys Val

130 135 140

gct gat cgg act ggc cac tcg cta ttg cac acc tta tat gga agg tct 480

Ala Asp Arg Thr Gly His Ser Leu Leu His Thr Leu Tyr Gly Arg Ser

145 150 155 160

ctg cga tat gat acc agc tat ttt gtg gag tat ttt gcc ttg gat ctc 528

Leu Arg Tyr Asp Thr Ser Tyr Phe Val Glu Tyr Phe Ala Leu Asp Leu

165 170 175

ctg atg gag aat ggg gag tgc cgt ggt gtc atc gca ctg tgc ata gag 576

Leu Met Glu Asn Gly Glu Cys Arg Gly Val Ile Ala Leu Cys Ile Glu

180 185 190

gac ggg tcc atc cat cgc ata aga gca aag aac act gtt gtt gcc aca 624

Asp Gly Ser Ile His Arg Ile Arg Ala Lys Asn Thr Val Val Ala Thr

195 200 205

gga ggc tac ggg cgc acc tac ttc agc tgc acg tct gcc cac acc agc 672

Gly Gly Tyr Gly Arg Thr Tyr Phe Ser Cys Thr Ser Ala His Thr Ser

210 215 220

act ggc gac ggc acg gcc atg atc acc agg gca ggc ctt cct tgc cag 720

Thr Gly Asp Gly Thr Ala Met Ile Thr Arg Ala Gly Leu Pro Cys Gln

225 230 235240

gac cta gag ttt gtt cag ttc cac cct aca ggc ata tat ggt gct ggt 768

Asp Leu Glu Phe Val Gln Phe His Pro Thr Gly Ile Tyr Gly Ala Gly

245 250 255

tgt ctc att acg gaa gga tgt cgt gga gag gga ggc att ctc att aac 816

Cys Leu Ile Thr Glu Gly Cys Arg Gly Glu Gly Gly Ile Leu Ile Asn

260 265 270

agt caa ggc gaa agg ttt atg gag cga tac gcc cct gtc gcg aag gac 864

Ser Gln Gly Glu Arg Phe Met Glu Arg Tyr Ala Pro Val Ala Lys Asp

275 280 285

ctg gcg tct aga gat gtg gtg tct cgg tcc atg act ctg gag atc cga 912

Leu Ala Ser Arg Asp Val Val Ser Arg Ser Met Thr Leu Glu Ile Arg

290 295 300

gaa gga aga ggc tgt ggc cct gag aaa gat cac gtc tac ctg cag ctg 960

Glu Gly Arg Gly Cys Gly Pro Glu Lys Asp His Val Tyr Leu Gln Leu

305 310 315 320

cac cac cta cct cca gag cag ctg gcc acg cgc ctg cct ggc att tca 1008

His His Leu Pro Pro Glu Gln Leu Ala Thr Arg Leu Pro Gly Ile Ser

325 330 335

gag aca gcc atg atc ttc gct ggc gtg gac gtc acg aag gag ccg atc 1056

Glu Thr Ala Met Ile Phe Ala Gly Val Asp Val Thr Lys Glu Pro Ile

340 345 350

cct gtc ctc ccc acc gtg cat tat aac atg ggc ggc att ccc acc aac 1104

Pro Val Leu Pro Thr Val His Tyr Asn Met Gly Gly Ile Pro Thr Asn

355 360 365

tac aag ggg cag gtc ctg agg cac gtg aat ggc cag gat cag att gtg 1152

Tyr Lys Gly Gln Val Leu Arg His Val Asn Gly Gln Asp Gln Ile Val

370 375 380

ccc ggc ctg tac gcc tgt ggg gag gcc gcc tgt gcc tcg gta cat ggt 1200

Pro Gly Leu Tyr Ala Cys Gly Glu Ala Ala Cys Ala Ser Val His Gly

385 390 395 400

gcc aac cgc ctc ggg gca aac tcg ctc ttg gac ctg gtt gtc ttt ggt 1248

Ala Asn Arg Leu Gly Ala Asn Ser Leu Leu Asp Leu Val Val Phe Gly

405 410 415

cgg gca tgt gcc ctg agc atc gaa gag tca tgc agg cct gga gat aaa 1296

Arg Ala Cys Ala Leu Ser Ile Glu Glu Ser Cys Arg Pro Gly Asp Lys

420 425 430

gtc cct cca att aaa cca aac gct ggg gaa gaa tct gtc atg aat ctt 1344

Val Pro Pro Ile Lys Pro Asn Ala Gly Glu Glu Ser Val Met Asn Leu

435 440 445

gac aaa ttg aga ttt gct gat gga agc ata aga aca tcg gaa ctg cga 1392

Asp Lys Leu Arg Phe Ala Asp Gly Ser Ile Arg Thr Ser Glu Leu Arg

450 455 460

ctc agc atg cag aag tca atg caa aat cat gct gcc gtg ttc cgt gtg 1440

Leu Ser Met Gln Lys Ser Met Gln Asn His Ala Ala Val Phe Arg Val

465 470 475 480

gga agc gtg ttg caa gaa ggt tgt ggg aaa atc agc aag ctc tat gga 1488

Gly Ser Val Leu Gln Glu Gly Cys Gly Lys Ile Ser Lys Leu Tyr Gly

485 490 495

gac cta aag cac ctg aag acg ttc gac cgg gga atg gtc tgg aac acg 1536

Asp Leu Lys His Leu Lys Thr Phe Asp Arg Gly Met Val Trp Asn Thr

500 505 510

gac ctg gtg gag acc ctg gag ctg cag aac ctg atg ctg tgt gcg ctg 1584

Asp Leu Val Glu Thr Leu Glu Leu Gln Asn Leu Met Leu Cys Ala Leu

515 520 525

cag acc atc tac gga gca gag gca cgg aag gag tca cgg ggc gcg cat 1632

Gln Thr Ile Tyr Gly Ala Glu Ala Arg Lys Glu Ser Arg Gly Ala His

530 535 540

gcc agg gaa gac tac aag gtg cgg att gat gag tac gat tac tcc aag 1680

Ala Arg Glu Asp Tyr Lys Val Arg Ile Asp Glu Tyr Asp Tyr Ser Lys

545 550 555 560

ccc atc cag ggg caa cag aag aag ccc ttt gag gag cac tgg agg aag 1728

Pro Ile Gln Gly Gln Gln Lys Lys Pro Phe Glu Glu His Trp Arg Lys

565 570 575

cac acc ctg tcc tat gtg gac gtt ggc act ggg aag gtc act ctg gaa 1776

His Thr Leu Ser Tyr Val Asp Val Gly Thr Gly Lys Val Thr Leu Glu

580 585 590

tat aga ccc gtg atc gac aaa act ttg aac gag gct gac tgt gcc acc 1824

Tyr Arg Pro Val Ile Asp Lys Thr Leu Asn Glu Ala Asp Cys Ala Thr

595 600 605

gtc ccg cca gcc att cgc tcc tac tga 1851

Val Pro Pro Ala Ile Arg Ser Tyr

610 615

<210>6

<211>616

<212>PRT

<213> Intelligent people

<400>6

Met Ser Gly Val Arg Gly Leu Ser Arg Leu Leu Ser Ala Arg Arg Leu

1 5 10 15

Ala Leu Ala Lys Ala Trp Pro Thr Val Leu Gln Thr Gly Thr Arg Gly

20 25 30

Phe His Phe Thr Val Asp Gly Asn Lys Arg Ala Ser Ala Lys Val Ser

35 40 45

Asp Ser Ile Ser Ala Gln Tyr Pro Val Val Asp His Glu Phe Asp Ala

50 55 60

Val Val Val Gly Ala Gly Gly Ala Gly Leu Arg Ala Ala Phe Gly Leu

65 70 75 80

Ser Glu Ala Gly Phe Asn Thr Ala Cys Val Thr Lys Leu Phe Pro Thr

85 90 95

Arg Ser His Thr Val Ala Ala Gln Leu Glu Asn Tyr Gly Met Pro Phe

100 105 110

Ser Arg Thr Glu Asp Gly Lys Ile Tyr Gln Arg Ala Phe Gly Gly Gln

115 120 125

Ser Leu Lys Phe Gly Lys Gly Gly Gln Ala His Arg Cys Cys Cys Val

130 135 140

Ala Asp Arg Thr Gly His Ser Leu Leu His Thr Leu Tyr Gly Arg Ser

145 150 155 160

Leu Arg Tyr Asp Thr Ser Tyr Phe Val Glu Tyr Phe Ala Leu Asp Leu

165 170 175

Leu Met Glu Asn Gly Glu Cys Arg Gly Val Ile Ala Leu Cys Ile Glu

180 185 190

Asp Gly Ser Ile His Arg Ile Arg Ala Lys Asn Thr Val Val Ala Thr

195 200 205

Gly Gly Tyr Gly Arg Thr Tyr Phe Ser Cys Thr Ser Ala His Thr Ser

210 215 220

Thr Gly Asp Gly Thr Ala Met Ile Thr Arg Ala Gly Leu Pro Cys Gln

225 230 235 240

Asp Leu Glu Phe Val Gln Phe His Pro Thr Gly Ile Tyr Gly Ala Gly

245 250 255

Cys Leu Ile Thr Glu Gly Cys Arg Gly Glu Gly Gly Ile Leu Ile Asn

260 265 270

Ser Gln Gly Glu Arg Phe Met Glu Arg Tyr Ala Pro Val Ala Lys Asp

275 280 285

Leu Ala Ser Arg Asp Val Val Ser Arg Ser Met Thr Leu Glu Ile Arg

290 295 300

Glu Gly Arg Gly Cys Gly Pro Glu Lys Asp His Val Tyr Leu Gln Leu

305 310 315 320

His His Leu Pro Pro Glu Gln Leu Ala Thr Arg Leu Pro Gly Ile Ser

325 330 335

Glu Thr Ala Met Ile Phe Ala Gly Val Asp Val Thr Lys Glu Pro Ile

340 345 350

Pro Val Leu Pro Thr Val His Tyr Asn Met Gly Gly Ile Pro Thr Asn

355 360 365

Tyr Lys Gly Gln Val Leu Arg His Val Asn Gly Gln Asp Gln Ile Val

370 375 380

Pro Gly Leu Tyr Ala Cys Gly Glu Ala Ala Cys Ala Ser Val His Gly

385 390 395 400

Ala Asn Arg Leu Gly Ala Asn Ser Leu Leu Asp Leu Val Val Phe Gly

405 410 415

Arg Ala Cys Ala Leu Ser Ile Glu Glu Ser Cys Arg Pro Gly Asp Lys

420 425 430

Val Pro Pro Ile Lys Pro Asn Ala Gly Glu Glu Ser Val Met Asn Leu

435 440 445

Asp Lys Leu Arg Phe Ala Asp Gly Ser Ile Arg Thr Ser Glu Leu Arg

450 455 460

Leu Ser Met Gln Lys Ser Met Gln Asn His Ala Ala Val Phe Arg Val

465 470 475 480

Gly Ser Val Leu Gln Glu Gly Cys Gly Lys Ile Ser Lys Leu Tyr Gly

485 490 495

Asp Leu Lys His Leu Lys Thr Phe Asp Arg Gly Met Val Trp Asn Thr

500 505 510

Asp Leu Val Glu Thr Leu Glu Leu Gln Asn Leu Met Leu Cys Ala Leu

515 520 525

Gln Thr Ile Tyr Gly Ala Glu Ala Arg Lys Glu Ser Arg Gly Ala His

530 535 540

Ala Arg Glu Asp Tyr Lys Val Arg Ile Asp Glu Tyr Asp Tyr Ser Lys

545 550 555 560

Pro Ile Gln Gly Gln Gln Lys Lys Pro Phe Glu Glu His Trp Arg Lys

565 570 575

His Thr Leu Ser Tyr Val Asp Val Gly Thr Gly Lys Val Thr Leu Glu

580 585 590

Tyr Arg Pro Val Ile Asp Lys Thr Leu Asn Glu Ala Asp Cys Ala Thr

595 600 605

Val Pro Pro Ala Ile Arg Ser Tyr

610 615

<210>7

<211>20

<212>DNA

<213> Intelligent people

<400>7

gctgagcaca ttgagtcacg 20

<210>8

<211>20

<212>DNA

<213> Intelligent people

<400>8

tggtacacct tggatgttgg 20

<210>9

<211>24

<212>DNA

<213> Intelligent people

<400>9

tagaaatcgc tgcgtacaga aaac 24

<210>10

<211>21

<212>DNA

<213> Intelligent people

<400>10

tgcttcctgc aaatgtgcta a 21

<210>11

<211>20

<212>DNA

<213> Intelligent people

<400>11

tgggaacaag agggcatctg 20

<210>12

<211>22

<212>DNA

<213> Intelligent people

<400>12

ccaccactgc atcaaattca tg 22

<210>13

<211>834

<212>DNA

<213> Intelligent people

<220>

<221>CDS

<222>(1)..(834)

<400>13

atg atc atc tta att tac tta ttt ctc ttg cta tgg gaa gac act caa 48

Met Ile IleLeu Ile Tyr Leu Phe Leu Leu Leu Trp Glu Asp Thr Gln

1 5 10 15

gga tgg gga ttc aag gat gga att ttt cat aac tcc ata tgg ctt gaa 96

Gly Trp Gly Phe Lys Asp Gly Ile Phe His Asn Ser Ile Trp Leu Glu

20 25 30

cga gca gcc ggt gtg tac cac aga gaa gca cgg tct ggc aaa tac aag 144

Arg Ala Ala Gly Val Tyr His Arg Glu Ala Arg Ser Gly Lys Tyr Lys

35 40 45

ctc acc tac gca gaa gct aag gcg gtg tgt gaa ttt gaa ggc ggc cat 192

Leu Thr Tyr Ala Glu Ala Lys Ala Val Cys Glu Phe Glu Gly Gly His

50 55 60

ctc gca act tac aag cag cta gag gca gcc aga aaa att gga ttt cat 240

Leu Ala Thr Tyr Lys Gln Leu Glu Ala Ala Arg Lys Ile Gly Phe His

65 70 75 80

gtc tgt gct gct gga tgg atg gct aag ggc aga gtt gga tac ccc att 288

Val Cys Ala Ala Gly Trp Met Ala Lys Gly Arg Val Gly Tyr Pro Ile

85 90 95

gtg aag cca ggg ccc aac tgt gga ttt gga aaa act ggc att att gat 336

Val Lys Pro Gly Pro Asn Cys Gly Phe Gly Lys Thr Gly Ile Ile Asp

100 105 110

tat gga atc cgt ctc aat agg agt gaa aga tgg gat gcc tat tgc tac 384

Tyr Gly Ile Arg Leu Asn Arg Ser Glu Arg Trp Asp Ala Tyr Cys Tyr

115 120 125

aac cca cac gca aag gag tgt ggt ggc gtc ttt aca gat cca aag caa 432

Asn Pro His Ala Lys Glu Cys Gly Gly Val Phe Thr Asp Pro Lys Gln

130 135 140

att ttt aaa tct cca ggc ttc cca aat gag tac gaa gat aac caa atc 480

Ile Phe Lys Ser Pro Gly Phe Pro Asn Glu Tyr Glu Asp Asn Gln Ile

145 150 155 160

tgc tac tgg cac att aga ctc aag tat ggt cag cgt att cac ctg agt 528

Cys Tyr Trp His Ile Arg Leu Lys Tyr Gly Gln Arg Ile His Leu Ser

165 170 175

ttt tta gat ttt gac ctt gaa gat gac cca ggt tgc ttg gct gat tat 576

Phe Leu Asp Phe Asp Leu Glu Asp Asp Pro Gly Cys Leu Ala Asp Tyr

180 185 190

gtt gaa ata tat gac agt tac gat gat gtc cat ggc ttt gtg gga aga 624

Val Glu Ile Tyr Asp Ser Tyr Asp Asp Val His Gly Phe Val Gly Arg

195 200 205

tac tgt gga gat gag ctt cca gat gac atc atc agt aca gga aat gtc 672

Tyr Cys Gly Asp Glu Leu Pro AspAsp Ile Ile Ser Thr Gly Asn Val

210 215 220

atg acc ttg aag ttt cta agt gat gct tca gtg aca gct gga ggt ttc 720

Met Thr Leu Lys Phe Leu Ser Asp Ala Ser Val Thr Ala Gly Gly Phe

225 230 235 240

caa atc aaa tat gtt gca atg gat cct gta tcc aaa tcc agt caa gga 768

Gln Ile Lys Tyr Val Ala Met Asp Pro Val Ser Lys Ser Ser Gln Gly

245 250 255

aaa aat aca agt act act tct act gga aat aaa aac ttt tta gct gga 816

Lys Asn Thr Ser Thr Thr Ser Thr Gly Asn Lys Asn Phe Leu Ala Gly

260 265 270

aga ttt agc cac tta taa 834

Arg Phe Ser His Leu

275

<210>14

<211>277

<212>PRT

<213> Intelligent people

<400>14

Met Ile Ile Leu Ile Tyr Leu Phe Leu Leu Leu Trp Glu Asp Thr Gln

1 5 10 15

Gly Trp Gly Phe Lys Asp Gly Ile Phe His Asn Ser Ile Trp Leu Glu

20 25 30

Arg Ala Ala Gly Val Tyr His Arg Glu Ala ArgSer Gly Lys Tyr Lys

35 40 45

Leu Thr Tyr Ala Glu Ala Lys Ala Val Cys Glu Phe Glu Gly Gly His

50 55 60

Leu Ala Thr Tyr Lys Gln Leu Glu Ala Ala Arg Lys Ile Gly Phe His

65 70 75 80

Val Cys Ala Ala Gly Trp Met Ala Lys Gly Arg Val Gly Tyr Pro Ile

85 90 95

Val Lys Pro Gly Pro Asn Cys Gly Phe Gly Lys Thr Gly Ile Ile Asp

100 105 110

Tyr Gly Ile Arg Leu Asn Arg Ser Glu Arg Trp Asp Ala Tyr Cys Tyr

115 120 125

Asn Pro His Ala Lys Glu Cys Gly Gly Val Phe Thr Asp Pro Lys Gln

130 135 140

Ile Phe Lys Ser Pro Gly Phe Pro Asn Glu Tyr Glu Asp Asn Gln Ile

145 150 155 160

Cys Tyr Trp His Ile Arg Leu Lys Tyr Gly Gln Arg Ile His Leu Ser

165 170 175

Phe Leu Asp Phe Asp Leu Glu Asp Asp Pro Gly Cys Leu Ala Asp Tyr

180 185 190

Val Glu Ile Tyr Asp Ser Tyr Asp Asp Val His Gly Phe ValGly Arg

195 200 205

Tyr Cys Gly Asp Glu Leu Pro Asp Asp Ile Ile Ser Thr Gly Asn Val

210 215 220

Met Thr Leu Lys Phe Leu Ser Asp Ala Ser Val Thr Ala Gly Gly Phe

225 230 235 240

Gln Ile Lys Tyr Val Ala Met Asp Pro Val Ser Lys Ser Ser Gln Gly

245 250 255

Lys Asn Thr Ser Thr Thr Ser Thr Gly Asn Lys Asn Phe Leu Ala Gly

260 265 270

Arg Phe Ser His Leu

275

Claims

1. A method of preparing a population of cells comprising adherent stem cells, the method comprising: obtaining a cell population having the cell characteristics of (a) and (b) shown below,

2. The method according to claim 1, wherein the relative expression amount of the NEFM gene of the cell population with respect to the expression amount of the SDHA gene is less than 0.02.

3. The production method according to claim 1 or 2, wherein the relative expression amount of the PLIN2 gene in the cell population with respect to the expression amount of the NEFM gene is 250 or more.

4. A cell population comprising adherent stem cells, which is a cell population having the cell characteristics of (a) and (b) shown below,

5. The cell population of claim 4, wherein the relative expression level of the NEFM gene relative to the expression level of the SDHA gene of the cell population is less than 0.02.

6. The cell population according to claim 4 or 5, wherein the relative expression amount of PLIN2 gene relative to the expression amount of NEFM gene in the cell population is 250 or more.

7. The cell population of any one of claims 4-6, wherein said adherent stem cells are from a fetal appendage.

8. A pharmaceutical composition comprising the population of cells of any one of claims 4-7, and a pharmaceutically acceptable vehicle.

9. A pharmaceutical composition comprising a population of cells according to any one of claims 4 to 7, and other administrable cells.

10. The pharmaceutical composition of claim 8 or 9, wherein 1 dose of adherent stem cells for a human is 1 × 10¹²Less than one/kg body weight.

11. The pharmaceutical composition according to any one of claims 8 to 10, wherein the pharmaceutical composition is an injectable preparation.

12. The pharmaceutical composition according to any one of claims 8 to 11, wherein the pharmaceutical composition is a preparation for transplantation of a cell pellet or a sheet-like structure.

13. The pharmaceutical composition according to any one of claims 8 to 12, which is a therapeutic agent for an immune-related disease.